Sample positioning and analysis system

ABSTRACT

Systems for positioning and/or analyzing samples such as cells, vesicles, cellular organelles, and fragments, derivatives, and mixtures thereof, for electrical and/or optical analysis, especially relating to the presence and/or activity of ion channels.

CROSS-REFERENCES

[0001] This application is a continuation-in-part of the following U.S.patent applications, which are incorporated herein by reference: Ser.No. 09/581,837, filed Jun. 16, 2000; and Ser. No. ______ , filed Sep.14, 2000, titled EFFICIENT METHODS FOR THE ANALYSIS OF ION CHANNELPROTEINS, and naming Christian Schmidt as inventor. U.S. patentapplication Ser. No. 09/581,837, in turn, claims priority from PCTPatent Application Serial No. PCT/IB98/01150, filed Jul. 28, 1998, whichclaims priority from Swiss Patent Application Serial No. 2903/97, filedDec. 17, 1997. Each of these patent applications is incorporated hereinby reference. U.S. patent application Ser. No. ______ , filed Sep. 14,2000, titled EFFICIENT METHODS FOR THE ANALYSIS OF ION CHANNEL PROTEINS,and naming Christian Schmidt as inventor, in turn, claims priority fromSer. No. 09/581,837, with priority claims as listed above, U.S.Provisional Patent Application Serial No. 60/232,365, filed Sep. 14,2000; Ser. No. 60/233,800, filed Sep. 19, 2000; and Ser. No. ______ ,filed Sep. 13, 2001, titled HIGH-THROUGHPUT PATCH CLAMP SYSTEM, andnaming Christian Schmidt as inventor. Each of these patent applicationsis incorporated herein by reference.

[0002] This application is based upon and claims the benefit under 35U.S.C. § 119 of the following U.S. provisional patent applications,which are incorporated herein by reference: Ser. No. 60/233,800, filedSep. 19, 2000, titled DESIGN OF HIGHLY INTEGRATED PHARMACEUTICALSCREENING CHIPS, and naming Christian Schmidt as inventor; and Ser. No.______ , filed Sep. 13, 2001, titled HIGH-THROUGHPUT PATCH CLAMP SYSTEM,and naming Christian Schmidt as inventor.

[0003] This application incorporates by reference in their entirety forall purposes the following U.S. Pat. Nos. 5,355,215, issued Oct. 11,1994; and No. 6,097,025, issued Aug. 1, 2000.

[0004] This application incorporates by reference in their entirety forall purposes the following patent applications: U.S. patent applicationSer. No. 90/708,905, filed Nov. 8, 2000; PCT Patent Application SerialNo. PCT/IB00/00095, filed Jan. 26, 2001; and PCT Patent ApplicationSerial No. PCT/IB00/00097, filed Jan. 26, 2001.

[0005] This application incorporates by reference in their entirety forall purposes the following U.S. patent applications: Ser. No.09/337,623, filed Jun. 21, 1999; Ser. No. 09/349,733, filed Jul. 8,1999; Ser. No. 09/478,819, filed Jan. 5, 2000; Ser. No. 09/596,444,filed Jun. 19, 2000; Ser. No. 09/710,061, filed Nov. 10, 2000; Ser. No.09/722,247, filed Nov. 24, 2000; Ser. No. 09/759,711, filed Jan. 12,2001; Ser. No. 09/765,869, filed Jan. 19, 2001; Ser. No. 09/765,874,filed Jan. 19, 2001; Ser. No. 09/766,131, filed Jan. 19, 2001; Ser. No.09/767,434, filed Jan. 22, 2001; Ser. No. 09/767,579, filed Jan. 22,2001; Ser. No. 09/767,583, filed Jan. 22, 2001; Ser. No. 09/768,661,filed Jan. 23, 2001; Ser. No. 09/768,765, filed Jan. 23, 2001; Ser. No.09/770,720, filed Jan. 25, 2001; Ser. No. 09/770,724, filed Jan. 25,2001; Ser. No. 09/777,343, filed Feb. 5, 2001; Ser. No. 09/813,107,filed Mar. 19, 2001; Ser. No. 09/815,932, filed Mar. 23, 2001; and Ser.No. 09/836,575, filed Apr. 16, 2001; and Ser. No. ______ , filed Aug.20, 2001, titled APPARATUS AND METHODS FOR THE GENERATION OF ELECTRICFIELDS WITHIN MICROPLATES, and naming James M. Hamilton as inventor.

[0006] This application incorporates by reference in their entirety forall purposes the following U.S. Provisional Patent Applications: SerialNo. 60/223,642, filed Aug. 8, 2000; Ser. No. 60/244,012, filed Oct. 27,2000; Ser. No. 60/267,639, filed Feb. 10, 2001; Ser. No. 60/287,697,filed Apr. 30, 2001; Ser. No., filed Aug. 2, 2001, titled pH PROBES FORCELL-BASED FLUORESCENCE ASSAYS, and naming Zhenjun Diwu, Jesse J. Twu,Guoliang Yi, Luke D. Lavis, and Yen-Wen Chen as inventors; and SerialNo. ______ , filed Aug. 31, 2001, titled KINETIC ASSAY FOR DETERMININGCALCEIN RETENTION IN CELLS, and naming Kelly J. Cassutt, Jesse J. Twu,and Anne T. Ferguson as inventors.

[0007] This application incorporates by reference in its entirety forall purposes the following publications: Richard P. Haugland, Handbookof Fluorescent Probes and Research Chemicals (6^(th) ed. 1996); andJoseph R. Lakowicz, PRINCIPLES OF FLUORESCENCE SPECTROSCOPY (2^(nd) Ed.1999).

FIELD OF THE INVENTION

[0008] The invention relates to systems for positioning and/or analyzingsamples. More particularly, the invention relates to systems forpositioning and/or analyzing samples such as cells, vesicles, cellularorganelles, and fragments, derivatives, and mixtures thereof, forelectrical and/or optical analysis, especially relating to the presenceand/or activity of ion channels.

BACKGROUND OF THE INVENTION

[0009] A variety of important biological processes occur at or withincell membranes. It therefore is not surprising that the biologicalfunction of membrane proteins has become an area of active research.Signal transduction processes in general, including nerve conduction,and neuroreceptors in particular have been shown to be influenced bypharmacologically active ingredients, making them obvious targets fordrug development.^(i) Ion channels and ion transporters also have beenshown to be an important class of therapeutic targets. In fact,interactions with ion channels have become a major potential source ofadverse effects when administering a therapeutic agent, leading the Foodand Drug Administration (FDA) and other government regulatory agenciesto require safety profiling of potential therapeutics against certainion channels.

[0010] This understanding of the interactions between potential drugsand cell membrane components is beginning to play a crucial role inmodern drug development. In view of the increasing number of knownreceptors and the rapidly growing libraries of potential pharmaceuticalingredients, there clearly is a need for highly sensitive screeningmethods that permit the analysis of a large number of differentsubstances with high assay throughput per unit time, otherwise known as“high throughput screening” (or “HTS”). In particular, there is a needfor automated and/or high throughput screening methods that are relevantto cell membrane components.

[0011] At present, relatively traditional methods are used for thescreening of pharmaceutical ingredients. Such methods include ligandbinding assays and receptor function tests that are performedseparately.^(ii) Although binding assays are relatively inexpensive, andamenable to high throughput, they require labeled high-affinity ligands,and generally are limited to assays for ligands that can competeeffectively for labeled ligand. Fluorescent or fluorogenic reagentsgenerally are compatible with high throughput assays, including theanalysis of ion channels using fluorescent calcium indicators, and theevaluation of membrane potential effects with potential-sensitive dyes.However, such reagents typically are not sensitive enough for singlecell measurements, and generally can provide only indirect measurementsof the membrane component of interest.

[0012] The patch clamp was introduced by Neher and Sakmann in the early1980s as a powerful technique for the direct study of drug effects onsingle receptors. In recognition of the strength of the method, Neherand Sakmann were awarded the Nobel prize in 1991. Classical patch-clampmethods often are used in conjunction with functional membrane receptorassays, including receptors coupled to G-proteins and ionchannel-forming receptors.^(iii) This method is highly specific andextremely sensitive: it can, in principle, be used to measure thechannel activity of individual receptor molecules. In doing so, glassmicropipettes with an opening diameter of typically 1-0.1 μm are pressedon the surface of a biological cell. The membrane surface that iscovered by the micropipette is called a “patch.” If the contact betweenthe glass electrode and the cell membrane surface is sufficientlyelectrically isolating, the ion flow over the membrane patch can bemeasured electrically with the aid of microelectrodes, one placed in theglass pipette and the other placed in the milieu opposite themembrane.^(iv) A key advantage of this electrophysiological method isthat it makes directly accessible the function of the correspondingchannel-forming proteins or receptors coupled to channel-formingproteins via the measured electrical characteristics of thechannel-forming proteins.

[0013] Unfortunately, several major limitations have preventedpatch-clamp technology from revolutionizing receptor science andpharmaceutical drug development. For example, to produce high qualityresults, the patch-clamp method requires a tremendous effort intechnical installation and highly qualified operators. Moreover, inaddition to being expensive, a standard patch-clamp setup may require along set-up time and have a high failure rate.

[0014] Thus, there is a need for a system for positioning and/oranalyzing cells that is rapid, facile, and suitable for multiarrayanalysis, such as the system provided by the invention.

SUMMARY OF THE INVENTION

[0015] The invention provides systems for positioning and/or analyzingsamples such as cells, vesicles, cellular organelles, and fragments,derivatives, and mixtures thereof, for electrical and/or opticalanalysis, especially relating to the presence and/or activity of ionchannels.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 is a schematic view of a system for positioning and/oranalyzing samples in accordance with aspects of the invention.

[0017]FIG. 2 is a cross-sectional side view of a substrate chip preparedfrom Si/SiO₂ in accordance with aspects of the invention.

[0018]FIG. 3 is a cross-sectional side view of a measurement systemhaving planar electrodes in accordance with aspects of the invention.

[0019]FIG. 4 is a cross-sectional side view of a measurement systemhaving point or wire electrodes in accordance with aspects of theinvention.

[0020]FIG. 5 is a cross-sectional side view of a measurement systemhaving open fluid compartments in accordance with aspects of theinvention.

[0021]FIG. 6 is a top view of a measurement system having multiplemeasurement sites in accordance with aspects of the invention.

[0022]FIG. 7 is a cross-sectional side view of the measurement system ofFIG. 6, taken generally along line 7-7 in FIG. 6.

[0023]FIG. 8 is a cross-sectional side view of a measurement systemhaving optical measurement aids in accordance with aspects of theinvention.

[0024]FIG. 9 is a partially cross-sectional partially schematic sideview of a measurement system for combined electrical/optical detection.

[0025]FIG. 10 is a contour plot of the electric potential adjacent anaperture in a substrate in accordance with aspects of the invention,computed by finite element method (FEM) simulation.

[0026]FIG. 11 is a plot of current versus time showing a decrease incurrent upon addition of Ca²⁺ to a final concentration of 4 mM followingthe docking of vesicles at a 7-μm aperture in the unmodified surface ofa suitable substrate.

[0027]FIG. 12AB is a pair of plots of current versus time showing thetime course of vesicle binding and the subsequent development ofmembranes with very high electrical insulation resistance for (A) a 4-μmaperture and (B) a 7-μm aperture in a poly-L-lysine-coated SiO₂substrate.

[0028]FIG. 13 is a plot of current versus time showing the passage ofindividual vesicles through a 7-μm aperture, as reflected influctuations in the plot recorded at a constant clamp voltage, V_(c), of−80 mV.

[0029]FIG. 14 is a plot of current versus time showing the time- andvoltage-dependent switching of alamethicin pores in a membrane producedon the substrate (C_(alamethicin)=0.1 μg/mL in 85 mM KCl) at negativepotentials.

[0030]FIG. 15AB is a pair of plots of current versus time showing thechanges in measured membrane resistance of a membrane produced on aSi/SiO₂ carrier chip after fusion with vesicles containing nAChR(nicotinic acetylcholine receptor). FIG. 15A shows the membraneresistance during accidental receptor openings in the absence of ligandsat 400 mM KCl and positive potentials. FIG. 15B shows the membraneresistance 150 seconds after the addition of the nAChR agonistcarbamylcholine (20 μm final concentration), where no receptor openingsare observed due to desensitization of the receptors.

[0031]FIG. 16 is a plot of current versus time showing the time courseof positioning, binding, and subsequent development of a tightelectrical seal for a Jurkat cell.

[0032]FIG. 17 is a series of plots of current versus time showing thecurrent flowing through the membrane of a Jurkat cell for the indicatedpositive and negative clamp voltages.

[0033]FIG. 18 is an analysis of the current flowing through the membraneof a Jurkat cell for a +60 mV clamp voltage showing (A) a representativeplot of current versus time, and (B) a histogram showing the relativelikelihood of the measured currents.

DETAILED DESCRIPTION

[0034] The invention provides systems such as single and multiaperturebiochips for positioning and/or analyzing membrane-bound samples, suchas cells, vesicles, cellular organelles, and/or portions thereof.Positioning a sample, as used here, generally comprises locating orplacing the sample at a preselected position, within the system,typically for subsequent analysis. Analyzing the sample generallycomprises detecting a presence or activity within the sample, while itis positioned at the preselected position, typically relating at leastin part to electrical properties of the sample.

[0035]FIG. 1 shows a representative system 30 for positioning and/oranalyzing samples in accordance with aspects of the invention. Thesystem includes a substrate 32, at least two fluid compartments 34 a,b,and at least two electrodes 36 a,b. (In some cases, for example, whereonly optical measurements are to be performed, the system may have onlya single fluid compartment and no electrodes.) The substrate comprises aseparating wall of electrically isolating material, and may include anaperture and/or window 38 and an associated adhesion surface 40 adjacentthe aperture to which samples 42 may be bind or be fixed using anysuitable mechanism. The fluid compartments generally comprise any regionor volume adapted to support a fluid adjacent the aperture. Theelectrodes generally comprise any mechanism for applying and/ormeasuring an electric potential and associated electric field across theaperture. In most embodiments, a first side of the substrate is used asa sample or measurement side, and a second side of the substrate is usedas a reference side, although these roles may be interchangeable. Themeasurement side is used to hold samples during positioning and/oranalysis, and typically includes adhesion surface 40, a measurementfluid compartment 34 a and a measurement electrode 36 a. The referenceside is used to complete the electric circuit, and typically includes areference fluid compartment 34 b and a reference electrode 36 b.Generally, despite their different names, the measurement electrode andthe reference electrode independently may be set to any suitablevoltage, including ground.

[0036] The system may include or be interfaced with one or moreauxiliary systems, including (1) a sample handling system 44 forintroducing, removing, and/or otherwise manipulating fluids and/orsamples, (2) an analysis system 46 for analyzing samples, particularlyby mechanisms other than direct electrical measurement, and/or (3) anincubation system for storing samples before and/or during assays,and/or (4) a cleaning system for cleaning substrates and/or other systemcomponents.

[0037] The system may be used for a variety of applications. Theseapplications may include automated and/or high-throughput patch-clampanalysis (e.g., for drug screening), portable biosensor analysis (e.g.,for environmental analytes), and so on. These applications also mayinclude the separation of cells or vesicles, the analysis of the sizesof cells or vesicles, the direct functional analysis of ionotropicmembrane proteins, for example, in ligand binding studies, and/or thepositioning of cells or vesicles for any suitable purpose, includingpurely optical investigations and/or microinjections, among others.Typically, a sample such as a cell or vesicle is introduced into themeasurement compartment and then is directed toward the aperture, forexample, using an electric force created by the two electrodes. Thesample contacts the adhesion surface, binds across the aperture, andforms an electrical seal with the aperture sufficient for performing anassay of interest. The effects of an applied voltage created by theelectrodes then may be studied, typically before and/or after exposureto a suitable assay condition The studies may be performed by measuringchanges in electrical properties across the aperture, such as current,resistance, or the like, and/or by measuring other changes in thesample, such as ion levels or the like.

[0038] The assay condition generally comprises any change of condition,optionally including a change in environmental condition, such as sampletemperature, but more typically including the addition of one or morereagents such as candidate drug compounds to the sample. The reagent maybe a chemical reagent, such as an acid, a base, a metal ion, an organicsolvent, or other substance intended to effect a chemical change in thesample. Alternatively, the reagent may have or be suspected to have abiological activity or type of interaction with a given biomolecule.Selected assay components may include membrane-active substances, suchas pore promoters, proteoliposomes, and/or membrane proteins. Selectedassay reagents also may include oligonucleotides, nucleic acid polymers,peptides, proteins, drugs, and other biologically active molecules.

[0039] The system may have one or more advantages over prior systems formeasuring electrical properties of cells and vesicles. First, the systemis relatively simple, both in the production of electrically insulatingpatch membranes and in the associated measurements. Thus, the system,alone or in combination with modern microtechnological methods, issuitable for use in automated and/or “high throughput screening” (HTS)applications. Second, the positioning and measuring capabilities of thesystem are well suited to the combination of electrical and opticalmeasurements, through which, on these planar membranes, obtained bymeans of the positioning process according to the invention, new,important information concerning membrane channels and receptors may beobtained.

[0040] The following sections describe various components andfunctionalities of the system, including (A) the substrate, (B) theapertures and windows, (C) the adhesion surfaces, including mechanismsfor achieving binding, (D) the fluid compartments, (E) the electrodes,(F) multiaperture systems, (G) the analysis system, (H) the samplehandling system, (I) the samples, (J) the sample positioning process,and (K) the measurement process, among others.

[0041] A. Substrates

[0042] The substrate generally comprises any surface or set of surfacescapable of separating two fluid compartments. The substrate typicallyincludes an aperture that passes through the substrate to connect thetwo fluid compartments and at least one adhesion surface adjacent theaperture for binding a sample such as a cell or vesicle for analysis.The substrate preferably is nonconductive (e.g., electricallyinsulating), thus reducing or eliminating electrical contact between thetwo fluid compartments, except through the aperture.

[0043] The substrate may be formed of any suitable material. Preferably,the substrate is nonconductive, inert (in the system), and no more thanslightly modifiable chemically. Exemplary materials include silicon(including silicon (Si) and silicon derivatives, such as silicon oxide(SO₂; silica) and silicon oxinitride (SiO_(x)N_(y))), glass, quartz,plastic, and so on. Among these, the silicon-based substrates haveseveral advantages. First, they are commercially available. Second, theyare easily processed, for example, so that they may be provided with anaperture and/or window, as described below in Example 1. Third, theyreadily may be coated or otherwise partially or completely covered withinsulating and/or adhesion-promoting materials. Such surface layersinclude layers of quartz, glass, and solid and/or gelatinous polymers,among others. Such surface layers also include plastomers andelastomers, such as polyimides, polymethylmethacrylates, polycarbonates,and silica gels (e.g. Sylgard). Such surface layers also may behomogeneous or inhomogeneous, where, for example, in the latter case,they may be applied as droplets.

[0044] The substrate may include two or more pieces or components, forexample, being constructed of a holder on which the material actuallyrelevant to membrane positioning and membrane binding is fastened orinto which this material is admitted, where this material for thepositioning, or alternatively the binding, of the membranes has at leastone aperture.

[0045] The substrate may be formed with any suitable geometry, subjectto the above limitations. However, preferably, the substrate is at leastsubstantially planar, and more preferably, the substrate ismicroscopically flat and molecularly relatively planar, particularly atthe adhesion surface.

[0046] Examples 1-7 below, among others, describe exemplary substrates,including materials, geometries, and relationships with other componentsof the system.

[0047] B. Apertures and Windows

[0048] The aperture and window(s) are the portions of the substrate mostimmediately involved in the positioning and/or analysis of samples.

[0049] The aperture generally comprises any opening or other passagethrough the substrate. This opening may include a hole, a gap, and/or aslit, among others, and may allow fluid contact between fluidcompartments positioned at opposite sides of the aperture. The aperturemay be capable of forming an electrical seal with a sample such as acell or vesicle that is sufficiently “tight” to use in a patch clampexperiment. Exemplary seals (depending on sample type and condition)have included >10 kΩ, >100 kΩ, >1 MΩ, >10 MΩ, >100 MΩ, >1 GΩ, >10GΩ, >100 GΩ, and even >1TΩ. Alternatively, or in addition, the aperturemay be capable of focusing an electric field with sufficient strength toposition a sample such as a cell or vesicle about the aperture. Theaperture may include a hole, a gap, and/or a slit, among others.

[0050] The aperture is characterized by a length L_(ap) and a diameterd_(aperture) The length is determined by the thickness of the substrateadjacent the aperture, generally ranging between about 3 μm and about1000 μm, and preferably ranging between about 100 nm and about 20 μm.The diameter of the aperture, as measured immediately adjacent thebinding surface, is influenced by a variety of different factors, whichmay urge toward either smaller or larger apertures. First, smallerapertures generally increase the quality of the electrical seal betweenthe aperture and the sample, up to a limit. In particular, to form atight electrical seal, the aperture should be smaller, preferablysignificantly smaller, than the size of the sample (i.e.,d_(aperture)<<d_(cell), d_(vesicle)), but larger, preferablysignificantly larger, than the lipids and other molecules present in thesample. Second, smaller apertures generally increase the mechanicalstability of the membrane across the aperture. In particular, the forcerequired to deflect a portion of membrane is inversely proportional tothe square of the radius of the portion being deflected (i.e.,proportional to the value of r_(M) ⁻²; see, e.g., Example 12), so thatthe selection of small (e.g., d_(aperture)<5 μm) apertures maysignificantly increase membrane stability, particularly relative to thetypical (e.g., d_(aperture)>100 μm) apertures used in conventional blacklipid membrane (BLM) systems. Third, smaller apertures generallyincrease the strength and focus of the electric field passing throughthe aperture, which is especially useful when positioning samples.Fourth, larger apertures generally reduce the access resistance,improving the quality of the voltage (or current) clamp, probably byeasing the physical access of the conduction ions. Based on thesefactors, the diameter of the aperture generally is less than about 15 to20 μm, usually is less than about 10 μm, preferably is less than about 7μm, and more preferably is less than about 5 μm. In particular, sizesbetween about 0.3 μm and about 7 μm may yield an outstanding probabilityand quality of sealing. Stated alternatively, the aperture preferablyshould have a diameter of no more than a few tens of percent, and morepreferably no more than about 30 percent, of the sample diameter. Thus,for cellular samples, which typically have a diameter of greater thanabout 20 μm, the aperture preferably should have a diameter of no morethan about 5-7 μm.

[0051] The window generally comprises a portion of the fluid compartmentadjacent and providing access to the aperture. The preferred size of thewindow is determined by factors analogous to those described above fordetermining the preferred size of the aperture. In brief, the diameterof the window preferably is less than about 1000 μm, and more preferablyis significantly smaller, being not more than about 100 μm.

[0052] Examples 1-7 below, among others, describe exemplary aperturesand windows, including geometries and relationships with othercomponents of the system.

[0053] C. Binding and Adhesion Surfaces

[0054] The adhesion surface generally comprises any surface or set ofsurfaces adjacent the aperture to which samples such as cells andvesicles may bind for analysis. The adhesion surface typically is atleast substantially planar over an area exceeding that of the boundportion of the sample but in some cases may be at least slightly concavein the direction of the sample. Thus, the adhesion surface may have anarea of at least about 25 μM² for a cell that is about 5 μm in (bound)diameter, at least about 100 μm² for a cell that is about 10 μm in(bound) diameter, and so on.

[0055] Binding, as used here, generally comprises any stable orsemi-stable association between a sample and an adhesion surface thatresults in an electrical seal between the sample and one or moreapertures that is sufficiently “tight” to allow the desired measurement.Binding may be mediated by any suitable mechanism, direct or indirect,including electrostatic interactions, covalent bonding, ionic bonding,hydrogen bonding, van der Waals interactions, and/orhydrophobic-hydrophilic^(v) interactions, among others. In general,binding may be facilitated by the appropriate selection, treatment,and/or modification of the substrate, the sample, the measurementmedium, or a suitable combination thereof.

[0056] Binding may be facilitated by appropriate selection of thesubstrate. Thus, preferred substrates typically include a relativelyflat or gently contoured binding surface adjacent the aperture ofinterest, so that cells or vesicles may bind to form an acceptable sealwith the aperture without unwanted or unnecessary deformation. Moreover,preferred substrates also typically include a modifiable bindingsurface, so that the surface may be treated as desired to promotebinding.

[0057] Binding also may be facilitated by appropriate treatment of thesubstrate, as suggested above. Thus, in some applications, the surfacemay be treated or otherwise modified so that electrostatic or, in givencases, hydrophobic, van der Waals or covalent binding of vesicles orcells, or -the corresponding membranes or membrane fragments, ispromoted. For example, the binding surface may be coated with anadhesion promoter, such as poly-L-lysine, poly-D-lysine, gelatin,collagen, laminin, fibronectin, proteoglycans, polyethylenimine,albumen, BIOMATRIX EHS (Nunc Nalge International), BIOBOND (ElectronMicroscopy Services, Inc.), and/or MATRIGEL (Becton-Dickinson), amongothers. Alternatively, or in addition, the binding surface is modifiedin a way that promotes molecule-specific binding, such as with avidinand/or biotin, or by modification with immobilized lectins.Alternatively, or in addition the binding surface (especially a siliconbinding surface) may be coated with an oxide or oxynitride layer.Alternatively, or in addition, the binding surface may be coated withlargely hydrophobic compounds such as Tocopherol. In some embodiments,an electrically charged surface may be generated by modification, inparticular, by means of polycations and/or silanes, for example,aminosilanes, or the substrate may have a coating or other surface layerwith an electrically charged surface. Microstructured silicon/siliconoxide or silicon/silicon nitride substrates are especially suitable forproviding a good electrostatic attraction, after being coated with asubstance lending the desired surface charge.^(vi) Finally, to improvethe quality and consistency of the surface characteristics, thesubstrate may be subjected to oxygen plasma cleaned and/or partially orcompletely hydrophylized before the modification of its surface and/orbefore its immediate use, in addition to or in lieu of the abovemodifications. In some aspects of the invention, unwantedhydrophylization/modification of the hydrophobic surface can be avoidedby using silicon nitride for the surface layer.

[0058] Binding also may be facilitated by appropriate selection and/ortreatment of the sample itself. Thus, the sample may include unsaturatedlipids or other compositions that increase the fluidity of its membrane,potentially enhancing membrane flexibility during binding and sealformation. Alternatively, or in addition, the sample may include chargedlipids or other compositions that increase the charge on the sample,potentially enhancing the ability of the sample to bindelectrostatically to substrate surfaces bearing an opposite charge. Forexample, for a positively charged substrate surface, the vesicle mightinclude negatively charged palmitoyl-oleoyl-phosphatidylglycerol (POPG).

[0059] In some cases, binding may be facilitated by interactions betweenspecific binding pairs (SBPs), where one member of the pair isassociated with the sample and the other member of the pair isassociated with the substrate. The interactions between members of aspecific binding pair typically are noncovalent, and the interactionsmay be readily reversible or essentially irreversible. An exemplary listof suitable specific binding pairs is shown in Table 1. TABLE 1Representative Specific Binding Pairs First SBP Member Second SBP Memberantigen antibody biotin avidin or streptavidin carbohydrate lectin orcarbohydrate receptor DNA antisense DNA enzyme substrate enzymehistidine NTA (nitrilotriacetic acid) IgG protein A or protein G RNAantisense RNA

[0060] Binding also may be facilitated by appropriate selection and/ortreatment of the measurement medium. For example, the medium may includebinding mediators that participate in or otherwise promote interactionsbetween the sample and substrate, for example, by forming cross-bridgesbetween the sample and substrate and/or by counteracting the effects ofbinding inhibitors associated with the sample, substrate, or medium. Thebinding mediators may act specifically, for example, by binding tospecific groups or molecules on the sample or substrate. Thus, biotinmight act as a specific binding mediator by binding to and cross-linkingavidin or streptavidin on the sample and substrate. The bindingmediators also may act less specifically, or nonspecifically, forexample, by binding to classes or categories of groups or molecules onthe sample or substrate. Thus, Ca²⁺ ions might act as a relativelynonspecific binding mediator by binding to and cross-linking negativecharges on the sample and substrate. Ca2+ ions are particularlyappropriate for mediating the binding of cells or vesicles containingnegative lipids and substrates containing negative surface charges, suchas SiO₂ substrates.

[0061] After binding, samples such as cells or vesicles may be brokenup, for example, by treatment with a hypotonic medium, such as purewater.

[0062] Examples 1-7, 10, and 11 below, among others, describe exemplaryadhesion surfaces, including materials, treatments, modifications,geometries, and the kinetics and efficacy of sample binding.

[0063] D. Fluid Compartments

[0064] The fluid compartments generally comprise any region or volumeadapted to support a fluid adjacent the aperture. The compartments mayperform several functions, including covering the sample, providing amedium through which the cell may be moved during positioning, and/orproviding a medium for establishing electrical contact between theelectrodes, among others. The compartments generally may have anysuitable volumes, but they typically have volumes between about 0.1 to40-100 μL. Thus, assays typically require only a limited amount ofsample, facilitating the analysis of effects of precious compounds.

[0065] The fluid compartments may be closed or open. A closedcompartment comprises a compartment that is at least substantiallybounded or delimited on all sides by a wall or other separating layer,exclusive of an input and/or output port. In contrast, an opencompartment comprises a compartment that is not bounded on at least oneside (i.e., over at least some solid angle) by a wall.

[0066] Closed compartments typically are physically confined, i.e.,bounded by some combination of the substrate, an electrode, and one ormore spacers, being established within voids and channels therein. Thespacers are used in many embodiments, particularly those involvingplanar electrodes, to establish and maintain the relative positions ofthe substrate and electrodes. Such spacers typically are formed of anelectrically isolating material, like the substrate. The spacers mayinclude channels disposed between the aperture and the electrode. Thesechannels, typically filled with a conductive solution, can serve as asample or reference chamber. It is beneficial if the reference chamberhas such small dimensions that the reference buffer solution may befixed therein by capillary forces, and/or forced therein by surfacetension (e.g., at the fluid/air interface in an open compartment).

[0067] Open compartments typically are free standing, i.e., not boundedin at least one, typically lateral, direction. Instead, the fluid may befixed between the substrate and electrode without other physicalboundaries by capillary forces and/or surface tension.

[0068] Examples 2-7 below, among others, describe exemplary fluidcompartments, including geometries, boundaries, and relationships withother components of the system.

[0069] E. Electrodes

[0070] The electrodes generally comprise any mechanism for creatingand/or modulating an electric potential across an aperture, particularlyfor use in positioning and/or analyzing samples.

[0071] The electrodes may be formed of any material capable of inducingcurrent flow through an aperture upon application of a physiologicalpotential. Suitable electrodes include silver, gold, and/or platinum,among others. Preferred electrodes include silver/silver chloride(Ag/AgCl) and/or platinum (Pt) redox electrodes.

[0072] The electrodes may be formed with any suitable geometry and bedisposed in any suitable arrangement, consistent with their performingtheir intended function(s). Preferred electrodes have planar,cylindrical, or point geometries. Preferred electrode arrangements aresymmetrical, with similar electrodes positioned at similar distances andorientations from each aperture on each side. Symmetrical electrodearrangements generally will create symmetrical electric fields.Typically, the electrodes are located opposite one another across asingle aperture, with each electrode reaching into at least onecompartment, or at least contacting a surface of it. The electrodescustomarily are located at a distance of about 0.5 to 3 mm, and usuallyabout 0.5 to 1 mm, from the substrate, although they can be closer orfarther in some embodiments. Depending on embodiment, the electrodes maybe attached directly to a recording carrier, or to a cartridge in whichthis carrier is packaged, and/or to a holder that is not in directcontact to the substrate.

[0073] The electrodes should be capable of creating an electricpotential sufficient to perform their function, for example, positioningand/or analyzing samples, without unduly disrupting the samples.Preferred electric potentials give rise to electric field intensities ofgreater than about 100 V/m, particularly adjacent the aperture. In thefollowing materials, one electrode is referred to as a measurementelectrode, p Examples 2-7 below, among others, describe exemplaryelectrodes, including geometries (e.g., planar, cylindrical, and point),materials (e.g., silver and/or platinum), and relationships with othercomponents of the system.

[0074] F. Multiaperture Systems

[0075] The invention provides multiaperture systems for positioningand/or analyzing samples. These systems include two or more apertures,which may be disposed at the same and/or separate sites. Aperturesdisposed at the same site may be used to study single samples at two ormore positions on the sample. In contrast, apertures disposed atseparate sites may be used to study two or more samples, sequentiallyand/or simultaneously, at one or more positions on each sample. Theproduction of multiaperture systems generally is straightforward,especially using silicon substrates and Ag/AgCl electrodes, both ofwhich are easily microstructurable. In particular, multiaperture systemsmay be produced from a single continuous substrate having two or moreapertures or by joining together two or more smaller substrates eachhaving one or more apertures. The latter approach may be less expensivefor substrates such as silicon with costs that increase faster thanarea.

[0076] Exemplary multiaperture systems employ a multiarray layout havinga plurality of separate measurement sites. In these systems, each siteincludes at least one aperture and fluid and electrical contact with atleast one fluid compartment and at least one electrode, respectively, oneach side of the aperture. The fluid compartments and electrodes on oneside of the substrate (the measurement side) generally are separated toallow independent recordings. However, the fluid compartments andelectrodes on the other side (the reference side) may be partially ortotally combined, because these components typically function merely toprovide a common electrical potential (e.g., ground). The samplegenerally may be positioned on either the measurement or the referenceside, although typically it is positioned on the measurement side sothat each fluid compartment independently can contain the same ordifferent types of samples. Thus, in these exemplary systems, severalapertures may be used on one substrate, and the measurements may beperformed over at least two apertures sequentially and/or in paralleland/or in such a manner that all or several electrodes on one side ofthe substrate have a common electrical potential, or, alternatively, arecombined to form one electrode. Similarly, more than two electrodes andmore than one aperture can be present in such a way that at least oneelectrode, for example, a reference electrode, serves the measurementvia more than one aperture, or the measurement arrangement can have asubstrate with more than one aperture and twice as many electrodes asapertures in such a way that one aperture always is located between twoelectrodes.

[0077] Measurement sites may be separated using any suitable mechanism,including hydrophilic/hydrophobic surface patterning, as describedbelow, and/or dividing the carrier surface into small compartment wells(e.g., by laminating a thin polydimethylsiloxane (PDMS) layer containingsmall holes to the carrier surface adjacent the aperture).

[0078] The multiaperture system generally may include any number ofmeasurement sites, positioned in any suitable arrangement, with anysuitable size or footprint, all consistent with forming electric fieldswithin each site to position and/or analyze samples. Preferredconfigurations may be selected based on utility and/or convenience.Thus, preferred systems may include features selected from standardmicroplates, so that the system may be used with standard microplateequipment, including handlers, washers, and/or readers, among others.These features may include a rectangular frame, with a major dimensionof about 125-130 mm, a minor dimension of about 80-90 mm, and a heightof about 5-15 mm, although other dimensions are possible. The frame mayinclude a base configured to facilitate handling and/or stacking, and/ora notch configured to facilitate receiving a cover. These features alsomay include 96, 384, 864, 1536, 3456, or 9600 measurement sites, amongothers, positioned on a rectangular or hexagonal array. Three exemplaryconfigurations that will fit as rectangular arrays within amicroplate-sized frame are listed in the following table: Arrangement ofPitch (mm) Density (/mm²) Number of Sites Sites Between Sites of Sites96  8 × 12 9 1/81 384 16 × 24 4.5 4/81 1536 32 × 48 2.25 16/81 

[0079] Here, pitch is the center-to-center site-to-site spacing, anddensity is the number of sites per unit area. These features also mayinclude the color of system components, particularly components in theoptical path in optical assays. For example, in fluorescenceapplications, system components preferably are made of opaque blackplastic to reduce background photoluminescence and/or “crosstalk,” wherecrosstalk is the transmission of light emitted in one site to adjacentsites where it may be detected. In contrast, in chemiluminescenceapplications, system components preferably are made of opaque whiteplastic to increase reflection of emitted light out of the site by thewhite surfaces while still reducing crosstalk.

[0080] Examples 5 and 6 below, among others, describe exemplarymultiaperture positioning and/or analysis systems, including additionalfeatures such as reference fiducials not described above.

[0081] G. Analysis System

[0082] The positioning and measurement system of the present inventionoptionally may be coupled to or integrated with an analysis system foranalyzing samples and sample components. The analysis system generallycomprises any mechanism for analyzing or otherwise characterizingsamples, qualitatively or quantitatively, other than by directelectrical measurement as used by the positioning and measurementsystem. The analysis system may require that the sample be separatedfrom the measurement system and/or from other sample components, asdescribed above. Alternatively, or in addition, the analysis system mayallow the sample to be studied in situ, without such separation.Generally, measurements made by the positioning and measurement systemand measurements made by the analysis system may be performedsimultaneously or sequentially, in any order or in any combination, inassociation with or independent of one another.

[0083] The analysis system may be based on any suitable analyticaltechnique, including spectroscopic, hydrodynamic, and imaging methods,among others, particularly those adaptable to high-throughput analysisof multiple samples. Preferred analysis systems are based on the opticalanalysis of samples, particularly luminescence-based optical analysis,but also absorption, scattering, circular dichroism, optical rotation,and imaging, among others. In luminescence analysis, light transmittedfrom the sample is detected and analyzed, and properties of the detectedlight are used to infer properties of the sample, including thepresence, size, shape, mobility, quantity, activity, and/or associationstate of selected components of the sample. In photoluminescence,including fluorescence and phosphorescence, the emission of light fromthe sample is induced by illuminating the sample with appropriateexcitation light. In chemiluminescence, the emission of light from thesample is induced by chemical reactions occurring within the sample. Theanalysis may involve measuring various properties of the detected light,including its intensity, lifetime, polarization, quantum yield, andStokes' shift, among others. The analysis also may involve using one ormore of these properties in techniques such as fluorescence intensity(FLINT), fluorescence polarization (FP), fluorescence resonance energytransfer (FRET), fluorescence lifetime (FLT), total internal reflectionfluorescence (TIRF), fluorescence correlation spectroscopy (FCS),fluorescence recovery after photobleaching (FRAP), and fluorescenceimaging, including confocal CCD observation, among others.

[0084] In luminescence assays, light typically is detected from aluminophore, i.e., a molecule or other species that emits luminescence.The luminophore may be endogenous or exogenous. Moreover, theluminophore may be the material of interest in the assay but morecommonly is simply a reporter that provides information about anothermaterial that is the true material of interest. In particular, theluminophore may be an exogenous molecule that reports on (1) membranepotential, (2) the presence or concentration of a target metal, such asCa²⁺, Mg²⁺, and Zn²⁺, (3) the presence or concentration of an inorganicion, such as Na⁺, K⁺, and Cl⁻, (4) pH, (5) reactive oxygen species,including nitric oxide, (6) ion channels, including Ca²⁺ channels, Na⁺channels, K⁺ channels, and Cl⁻ channels, (7) signal transduction, (8)cell viability, and (9) endocytosis and exocytosis, among others.Suitable luminophores for reporting on this and other information aredescribed in Richard P. Haugland, Handbook of Fluorescent Probes andResearch Chemicals (6^(th) ed. 1996), which is incorporated herein byreference.

[0085] The combination of optical technologies with the patch-clamptechnologies presented here permits, for the first time, the distinctionor resolution of ligand binding events and channel activities, amongothers. In this way, for example, important information regarding thestabilization of changes in receptor conformation through ligand bindingand/or the functional variation in ligand binding sites in receptors maybe obtained.^(vii) Such information is potentially important forunderstanding the particular mode of action of individual agonists andantagonists, and thus exhibits great promise for future drugdevelopment.

[0086] The optical analysis system typically will include a lightsource, a detector, and one or more optical relay structures fordirecting excitation light from the light source to the sample and fordirecting emission light from the sample to the detector. However, thelight source and excitation optical relay structure are optional duringanalysis utilizing chemiluminescence methods. The optical analysissystem may use epi- and/or trans-detection schemes, involvingilluminating off of and/or through the sample, respectively.

[0087] Exemplary optical analysis systems, and components thereof, aredescribed below under Examples and in the various patents, patentapplications, and other materials listed above under Cross-Referencesand incorporated herein by reference. Preferred optical analysis systemsare described in the following materials, which are incorporated hereinby reference: U.S. Pat. No. 5,355,215, issued Oct. 11, 1994; U.S. Pat.No. 6,097,025, issued Aug. 1, 2000; U.S. Provisional Patent ApplicationSerial No. 60/267,639, filed Feb. 10, 2001; and Joseph R. Lakowicz,Principles of Fluorescence Spectroscopy (2^(nd) ed. 1999).

[0088] The combined system is suitable for simultaneous and/orsequential electrical and optical (e.g., photoluminescence)measurements. Suitable apparatus may include a planar, and verticallyeasily realizable, optically transparent structure, for example, withthe use of planar pointed electrodes, or alternatively point electrodesdisposed outside of the vertical lines going through the aperture. Thisallows not only fluorescence or electrical analysis of single cells (ormembranes) but also the combined optical-electrical observation of cells(or membranes), particularly in response to exposure to externallyapplied substances, such as potential medical drugs. This revolutionaryapproach allows significantly more efficient selection of new drugs andthe elucidation of their molecular action.

[0089] Example 7 below, among others, describes an exemplary analysissystem. Additional examples are described in the various patents andpatent applications listed above under Cross-References and incorporatedherein by reference.

[0090] H. Sample-handling System

[0091] The positioning and measurement system according to the inventionoptionally may be coupled to or integrated with a sample-handling systemfor adding, manipulating, exchanging, and/or removing samples and samplecomponents, including cells and vesicles, sample media, and compoundsand reagents, such as candidate modulators and/or other analytes. Thesample-handling system may add samples such as cells or vesicles toarbitrary compartments, convey liquid into and/or out of arbitrarycompartments, and/or exchange samples and/or liquid between arbitrarycompartments, among others. The sample-handling system also may separatesamples, or sample components, in particular using capillaryelectrophoresis (CE) and/or high-pressure liquid chromatography (HPLC),and serve the analysis of the separated substances, or it can beprovided with means that serve the continuous or regular testing of thestate of the liquid in the compartments as well as with means forretroactive regulation according to preset filling parameters. Becauseit is reasonable, according to the analysis strived for, to bring themembrane into contact with measurement solution on both sides, theaddition of a substance to be investigated obviously can be done on theside customarily serving as the reference side. The sample-handlingsystem may be multiplexed to interact with several substrates and/orwith a multiaperture substrate, among others.

[0092] The sample-handling system may be based on any suitablemechanism, including tubes, pumps, hydrostatic pressure differentials,electro-osmotic processes, piezo drop-on-demand processes, ink-jetprocesses, contact transfer processes, temperature-controlled processes,and/or mechanical displacement, among others. In some embodiments,fluids such as reference buffers may be introduced into a pasty gel,whereby an exchange of the liquid lying outside the gel is possiblewithout changing the composition of the reference buffer stored in thegel. Suitable gels include agarose and polyacrylamide.

[0093] Example 2 below, among others, describes an exemplary samplehandling system. Additional examples are described in the variouspatents and patent applications listed above under Cross-References andincorporated herein by reference, including U.S. patent application Ser.No. 09/777,343, filed Feb. 5, 2001; and U.S. Provisional PatentApplication Serial No. 60/267,639, filed Feb. 10, 2001.

[0094] I. Samples

[0095] The sample generally comprises any species having a membrane orother surface capable of forming a seal with an aperture sufficient forperforming electrical measurements such as patch clamp experiments. Thesample may include cells, vesicles, cellular organelles, membrane-boundviruses, and fragments, derivatives, and mixtures thereof.

[0096] Biological samples may include or be derived from (1) eukaryoticcells, i.e., cells with a nucleus, including cells from plants, animals,fungi, yeast, and protozoans, or enucleated derivatives thereof; (2)prokaryotic organisms, including bacteria and archaebacteria; (3)viruses; (4) organelles or extracts, such as nuclei, mitochondria,endosomes, the Golgi apparatus, peroxisomes, lysosomes, endoplasmicreticulum, chloroplasts, axons, and dendritic processes, among others;and (5) gametes, including eggs and sperm. These cells and othermaterials may be obtained from any suitable source, including cellcultures, patient samples, and tissues, among others. These cells alsomay be subjected to any suitable treatments to alter membraneproperties, for example, to introduce a novel or modified ion channel,among others. These treatments may include genetic modification by anysuitable method, including chemical treatment, irradiation,transfection, infection, and/or injection, among others.

[0097] Vesicles and other synthetic samples may include or be derivedfrom (1) unilamellar vesicles, (2) multilamellar vesicles, (3) smallvesicles (having diameters less than about 1000 nm), (4) large vesicles(having diameters greater than about 1000 nm), (5) monodispersevesicles, and (6) polydisperse vesicles. These vesicles may be formedfrom any suitable lipid(s) and/or protein(s) using any suitabletechnique. Exemplary lipids include DLPC, DMPC, DPPC, DSPC, DOPC, DMPE,DPPE, DOPE, DMPA, DPPA, DOPA, DMPG, DPPG, DOPG, DMPS, DPPS, and DOPS,among others.

[0098] Examples 8 and 15-17 below, among others, describe exemplarysamples, including vesicle and cell samples.

[0099] J. Sample Positioning

[0100] The electrical, optical, and/or other analysis of samplesgenerally is preceded by a positioning step, in which the sample isdirected to or otherwise located at the adhesion surface. Samplepositioning generally occurs in two sequential substeps: (1) a first(prepositioning) substep, in which the sample is introduced to themeasurement compartment, and (2) a second (micropositioning) substep, inwhich the sample is brought into proximity or actual contact with theadhesion surface. These steps may be performed robotically, at leastsubstantially without direct human involvement or intervention.

[0101] The prepositioning substep generally involves introducing thesample to the measurement compartment, preferably in a manner thatfacilitates subsequent binding of the sample to the adhesion surface.Thus, the sample may be introduced generally above the adhesion surface(or associated aperture), so that it is directly between the electrodes,if they are symmetrically arranged, and so that gravity will tend topull it straight down toward the aperture. Alternatively, or inaddition, the sample may be introduced relatively close to the adhesionsurface, and/or with an initial velocity toward the adhesion surface,among others.

[0102] The micropositioning step generally involves bringing the sampleinto proximity or actual contact with the adhesion surface, once thesample is in the measurement compartment. Generally, samples may bemicropositioned using any suitable force or other mechanism, includingsedimentation (e.g., “1g-sedimentation” under the influence of gravity),electromagnetic forces (e.g., electrophoresis, electro-osmosis, and thelike), optical forces (e.g., optical tweezers), fluid-mediated forces(e.g., pressure, vacuum, flow, diffusion, and the like), and/or manualforces. Alternatively, or in addition, the cells may be positioned byfluid flow from the sample compartment to the reference compartment by ahydrostatic pressure difference or by a difference in surface tensionbetween the two compartments. Preferably, samples are micropositionedusing electromagnetic forces, specifically, field focusing of anelectric field created by applying a potential across the twoelectrodes. In brief, this technique exploits the field focusing thatoccurs adjacent the aperture, creating an electric force that, at leastadjacent the aperture, points in all positions toward the aperture, witha strength that increases with proximity to the aperture.

[0103] Examples 2 and 9 below, among others, describe exemplary methodsfor prepositioning and micropositioning samples, respectively.

[0104] K. Measurement Process

[0105] The measurement process provided by aspects of the inventionallows in particular the measurement of ion channel flows in a reliableand reproducible manner, often with a high signal-to-noise ratio. Theseabilities reflect the precise positioning and electrically tight bindingof cells, vesicles, cellular organelles, and/or membranes ofcorresponding origin, at microstructured apertures in a planarsubstrate. This electrically tight binding may be achieved at least inpart by strong interactions between the surface of the substrate and thesurface of the bound membrane, such as strong electrostatic attractions.

[0106] The electrical characteristics of transmembrane ion channels orionotropic receptors may be characterized using “voltage-clamptechnologies,” such as classical voltage-clamp, patch-clamp, and oocytevoltage-clamp, among others.^(viii) Specifically, an electricalpotential difference is applied across the membrane containing therelevant ion channel(s), and, simultaneously, the current necessary tomaintain this difference is analyzed. The relationship between thevoltage and current may be expressed mathematically using Ohm's Law,which states that V=IR, or equivalently, I=V/R, where V=voltage,I=current, and R=resistance. The current provides insight into membraneelectrical properties, such as its conductivity, and therefore insightinto the conformation state of the channel-forming protein (e.g., open(passing ions) or closed (blocking ions)). Thus, the current may be usedto analyze voltage dependencies, ligand binding events, and so on.

[0107] The ability of current measurements to yield meaningful data in apatch clamp or other electrophysiology experiment is dependent onensuring that the measured current reflects ion flow through the sample(e.g., through ion channels in the membrane) and not through othercomponents of the system. In particular, to obtain acceptable or bettersignal-to-noise ratios, it typically is desirable to ensure that themeasured current includes no more than a ten or twenty percentcontribution from unintended sources (e.g., that sources of noise lieunder the signals to be measure by approximately the factor of five orten). Unfortunately, the ion flow through ionotropic membrane proteinswith 0.1 to 50 pA at a −60-mV membrane potential is in general verysmall, so that leakage currents occurring essentially between themembrane and its fastening quickly may become significant, representinga principal problem in all voltage-clamp technologies.

[0108] The problem of leakage currents can be solved in a variety ofways. For example, enlarging the aperture and thereby the patch ofmembrane to be analyzed may reduce the contribution of leakage currentsto the total signal, because the area of the membrane patch and so theintended signal will grow as the radius of the aperture squared, whilethe circumference of the membrane and so the leakage current will growmerely as the radius. Unfortunately, increasing the size of the membranemay lead to a loss in specificity, particularly in biological systems,because more channels and more types of channels will be in the membranearea analyzed. Then, in general, an unambiguous or completelyartifact-free statement, for example, in the case of the addition ofligands, may no longer be possible.

[0109] Establishing and maintaining a very high seal between themembrane and aperture also may reduce the contribution of leakagecurrents. This invention uses this principle, at least in part. Toimplement the seal, a planar substrate chip having a surface that isstrongly adhesive for cells and vesicles is used. This chip separatesthe two compartments clamped at different potentials during themeasurement, where a (sub)micrometer-sized aperture is located in itsmiddle. This aperture is filled with reference buffer solution andelectrically tightly sealed during current measurements by strongbinding of cells and vesicles to the surface. This electrically tightbinding may permit the measurement of very small ion flows (e.g., downto at least about 0.1 pA) and, concomitantly, the plotting of membraneresistance with a good or better signal-to-noise ratio.

[0110] Further (mechanical) stability may be derived from usingcapillary forces to fill and store the reference and/or measurementbuffers. In particular, unbounded or open fluid compartments mayexperience fewer disturbances (e.g., due to temperature differentials)of the membrane due to hydrostatic pressure than closed systems.

[0111] The measurement systems described here may be used for a varietyof applications, some of which are described below in Example 16,including “perforated-patch,” “whole-cell,” and “inside-out” patch clamptechniques. For example, measurement systems of the planar type areparticularly well suited, due to the short diffusion times associatedtherewith, to the use of “perforated-patch” techniques.^(IX) In thesetechniques, an electrical connection to the interior of the cells(cytosol) is achieved by permeabilization of the area of the membranesuspended across the aperture with pore-forming antibiotics. Anadvantage of this technique is that it does not require washing out thecytosol with measurement buffer solution for simultaneous electricalaccess. In particular, a pore former such as, for example, amphotericinB or nystatin can be added to the reference compartment, after abiological cell, or, under special circumstances, a vesicle (if itsmechanical stability is sufficiently high) is bound to the upper side ofthe aperture. In doing so, the rate of perforation of the membrane patchover the aperture is significantly greater than in comparable standardpatch-clamp techniques.

[0112] For example, measurement systems of the planar type also areparticularly well suited to the use of “whole-cell” techniques. In thesetechniques, an electrical connection to the cytosol is achieved bydestroying the membrane patch, for example, using a voltage pulse. Thisdestruction, in turn, may facilitate the simple addition of largerproteins into the cytoplasm, via the reference solution, again becausethe planar layout of the measurement system allows significantly fasterdiffusion of large macromolecules into the cytosol or the interior of avesicle than comparable standard whole-cell techniques.^(x)

[0113] The system facilitates the addition and/or exchange of varioussystem components, including solutions and/or substances, as suggestedabove. For example, in some applications, the measurement solution, thereference solution, or both solutions may be replaced by anothersolution. Alternatively, or in addition, a substance to be analyzed maybe added to the solution on the measurement and/or reference side. Thesubstance may include a pore former that can be added to one or bothcompartments with the aim of increasing the electrical conductivity, or,alternatively, the permeability of the membrane with respect to certainions. The substance also may include detergent-solubilized proteins orproteoliposomes of arbitrary size, with the aim of fusing them to themembrane over the aperture and thereby making arbitrary membraneproteins contained therein accessible to electrical or opticalmeasurements. The fusion of proteoliposomes is described in detail inU.S. patent application Ser. No. ______ , filed Sep. 14, 2001, titledEFFICIENT METHODS FOR THE ANALYSIS OF ION CHANNEL PROTEINS, and namingChristian Schmidt as inventor.

[0114] Examples 12-16 below, among others, describe exemplary resultsobtained from various electrical measurements on vesicle andcell-derived membranes.

EXAMPLES

[0115] The following examples describe selected aspects and embodimentsof the invention. These examples are included for illustration andshould not be interpreted as restricting, limiting, or defining theentire scope of the invention. Additional examples are described in thefollowing patent applications, which are incorporated herein byreference: U.S. Provisional Patent Application Serial No. ______ , filedSep. 13, 2001, titled HIGH-THROUGHPUT PATCH CLAMP SYSTEM, and namingChristian Schmidt as inventor; and U.S. patent application Ser. No.______ , filed Sep. 14, 2001, titled EFFICIENT METHODS FOR THE ANALYSISOF ION CHANNEL PROTEINS, and naming Christian Schmidt as inventor.

Example 1

[0116] Substrate Chip

[0117] This example, illustrated in FIG. 2, describes an exemplarySi/SiO₂ chip substrate 50 for use in positioning and/or studying cells,vesicles, and the like, in accordance with aspects of the invention.

[0118] The substrate includes a body 52, a surface layer 54, a window56, and an aperture 58. The body comprises an at least substantiallyplanar, commercially available silicon wafer. The surface layercomprises a silicon oxide or silicon oxynitride layer formed adjacentone or more sides of the body. In this embodiment, the surface layer hasa thickness of at least about 50 to 200 nm and provides at least oneadhesion surface 60 a,b capable of binding cells, vesicles, and/or othersamples. The window and aperture comprise openings through the body andsurface layer, respectively. These openings are at least substantiallyconcentrically aligned, with dimensions sufficient to allow fluidcontact between opposite sides of the substrate.

[0119] The substrate may be produced using any suitable method,including photolithography or, for apertures having diameters of lessthan about 1.5 μm, electron beam lithography. These methods may involveanisotropic etching of the silicon in a medium containing KOH, as wellas reactive ion etching of the silica layer.

[0120] In alternative embodiments, the substrate may include a bodyand/or a surface layer having a different geometry and/or formed ofdifferent materials. In addition, the window may be absent, or thewindow and the aperture both may be openings in the body, particularlyin embodiments lacking a surface layer.

Example 2

[0121] Measurement System with Planar Electrodes

[0122] This example, illustrated in FIG. 3, describes an exemplarymeasurement system 70 having planar electrodes, in accordance withaspects of the invention.

[0123] The measurement system includes a substrate 72, at least twofluid compartments 74 a,b, at least two redox electrodes 76 a,b, andoptionally at least four spacers 78 a-d. In this embodiment, all ofthese components are at least substantially planar; however, in otherembodiments, one or more of these components may have a differentgeometry. Generally, samples may be introduced into either compartment,and measurements may be performed with the system in any orientation.However, to simplify the description, the top fluid compartment 74 a andtop electrode 76 a (as drawn) are referred to here as the measurementcompartment and measurement electrode, and the bottom fluid compartment74 b and the bottom electrode 76 b (as drawn) are referred to as thereference compartment and the reference electrode.

[0124] The substrate is used to support cells, vesicles, and othersamples for electrical analysis. The substrate includes a body 80, awindow 82, and an aperture 84 connecting the two fluid compartments. Thesubstrate further includes at least one adhesion surface 86 a,bpositioned adjacent one or both ends of the aperture for binding cells,vesicles, and/or other samples. An exemplary substrate is described inmore detail in Example 1.

[0125] The fluid compartments are used to support fluids such aselectrolyte solutions or growth media in apposition to the substrate andaperture. The compartments are formed by apertures or voids in thesubstrate, spacers, and/or electrodes.

[0126] The electrodes are used to apply and/or measure an electricpotential and associated electric field across the aperture. Measurementelectrode 76 a comprises a 0.8-mm thick chlorinated square (e.g., 4×4mm²) or annular (e.g., d=2 mm) silver (Ag) plate, preferably having afrustoconical or funnel-shaped opening 88 (e.g., d_(min)=0.4 to 1 mm).The measurement electrode is positioned at least substantially parallelto the surface of the substrate, preferably at a distance of up to about1 mm from the surface of the substrate. The measurement electrodefurther is positioned so that opening 88 is at least substantiallyconcentrically positioned above aperture 84. Reference electrode 76 bcomprises a 2-mm thick square silver plate (e.g., 20×20 mm²), whichpreferably has a purity greater than about 99.98% silver. The preferredsilver/silver chloride (Ag/AgCI) reference electrode may be producedduring manufacture of the system, for example, (1) by exposing thereference compartment to a molecular Cl₂ gas, typically while applying apotential to the electrode, or (2) by filling the reference compartmentwith 1 M HCl, and then chlorinating the exposed silver for 90 secondsunder a 0.8-V potential. The substrate is mounted, after its undersideis wetted with buffer solution, over the reference compartment filledwith reference buffer solution. In this embodiment, the referenceelectrode functions to support and maintain other components of thesystem.

[0127] The spacers may be used for several functions, including (1)separating the substrate and electrodes, (2) forming the fluidcompartments, and (3) contributing to the structure of a feed opening 90used to introduce samples to the sample compartment. Specifically, firstand second spacers 78 a,b are positioned about the measurementelectrode. These spacers include openings 92 a,b that may be alignedconcentrically with opening 88 in the measurement electrode and aperture84 in the substrate to form a “feed opening” for introducing samplesinto the system. The feed opening may be of arbitrary form; typically,however, it is elliptical, in particular circular, with a preferreddiameter of about 0.2 to 2 mm, and a more preferred diameter of about0.5 to 1 mm, to facilitate its concentric alignment with the aperture. Athird spacer 78 c is positioned between the measurement electrode andthe substrate to form an insulating barrier between these two elements.This spacer, preferably formed from silicone (e.g., Sylgard 184, DowComing, USA), includes a ring-like opening 92 c that again may bemounted concentrically about aperture 84, for example, with a radius rof about 1 mm. The ring-like opening forms, together with the meniscusthat forms between the chip and measurement electrode, the samplechamber (sample compartment) for the addition of cells, vesicles, and/ormeasurement solution. Finally, a fourth spacer 78 d is positionedbetween the reference electrode and the substrate to forming aninsulating barrier between these two elements. This spacer, preferablyformed as a 0.5 to 2 mm-thick silicon rubber seal (e.g., Sylgard),includes a channel or chamber 92 d having dimensions of about 1 mm inwidth and less than about 6 mm in length. The spacer may be imprintedand, if filled with buffer solution, produce contact between theaperture, or alternatively the membrane, and the reference electrode.

[0128] The measurement system may be configured or adapted to facilitatethe addition, positioning, and/or analysis of samples. Thus, the setuppreferably has means on one or both sides of the substrate that makepossible an addition of liquid, a storage of liquid, and, in givencases, an exchange of liquid, as well as the addition of cells,vesicles, or other cellular organelles, or parts of the same, betweenthe substrate and the electrode(s). For example, during measurement, ormembrane production, a small (e.g., 5 to 10 μL) volume of measurement orvesicle solution (e.g., a cell suspension) may be added (e.g., bypipette) directly to the feed opening, the window, and/or the aperture,on the measurement side of the substrate or on the upper side of themeasurement electrode. The aperture preferably has a diameter such that,when a voltage differential exists over the chip, an inhomogeneouselectrical field, mediated by the electrodes, is set up around theaperture. This field may increase in magnitude near the aperture, suchthat samples can be moved electrophoretically toward the aperture.Furthermore, the substrate preferably includes at least one surface 94a,b, on one or both sides of the aperture, that is attractive forbiological membranes, permitting the molecule-specific and/ormultivalent ion-mediated binding of cells, vesicles, membrane fragments,and/or cellular organelles. The surface of the substrate further may bestructured to create hydrophilic and hydrophobic areas, with thehydrophilic area preferably positioned around the aperture.

Example 3

[0129] Measurement System with Point or Wire Electrodes

[0130] This example, illustrated in FIG. 4, describes an exemplarymeasurement system 110 having point or wire electrodes, in accordancewith aspects of the invention.

[0131] The measurement system includes a substrate 112, two fluidcompartments 114 a,b, and two point or wire electrodes 116 a,b. Thesubstrate and fluid compartments are used to support samples and fluids,respectively, as described above. The substrate includes a body 118, awindow 120, and an aperture 122 connecting the two fluid compartments.The substrate further may be surface modified and/or fastened to aholder, including a glass or Teflon holder. The electrodes are used toapply an electric potential and associated electric field across theaperture, also as described above. Here, the electrodes comprise thechlorinated end surfaces 124 a,b of two silver wires 126 a,b, or,alternatively, two silver electrodes, disposed above and below thesubstrate. The electrodes preferably have diameters between about 0.1and 2 mm and a relative separation of about 4 mm. In some embodiments,the electrodes may be provided with a protective outer layer 128 a,bthat covers and protects the outside surface of the electrodes, exceptat the end surfaces.

[0132] The measurement system may be used for positioning and/oranalyzing samples. In an exemplary approach, sample medium is added toboth sides of the substrate, and held between the substrate andelectrode by capillary forces. Next, the offset is calibrated, and asuitable voltage is applied (typically, V=−60 to −100 mV). Then, cellsor vesicles are added to an appropriate (e.g., modified) side of thesubstrate, and cell binding and/or membrane formation are pursued withthe aid of a change in the electrical parameters. Finally, theproperties of ion channels or other membrane components are studiedusing suitable electrophysiology methods. Throughout, the addition orexchange of samples and/or sample media may be performed using a samplehandling system, as described above, such as a pipette or tube mountednear the aperture.

Example 4

[0133] Measurement System Having Open Fluid Compartments

[0134] This example, illustrated in FIG. 5, describes an exemplarymeasurement system 150 having open fluid compartments, in accordancewith aspects of the invention.

[0135] The measurement system includes a substrate 152, at least twofluid compartments 154 a,b, and at least two electrodes 156 a,b. Thesecomponents perform at least substantially the same functions as theirnamesakes in Examples 2 and 3.

[0136] The substrate comprises an insulating silicon chip 158. Thesubstrate may include a groove that is closed by a thin silicon nitride(Si₃N₄) 160/silicon oxide (SiO2) 162 diaphragm containing a smallaperture 164 having a diameter that usually is less than about 20 μm.The substrate further may include a surrounding insulating layer 166,for example, thermally grown silicon oxide, to reduce systemcapacitance. The surface of the substrate may be treated to promote thetight adhesion of cell or vesicle-associated lipid bilayers, forexample, by (1) physisorption of poly-L-lysine (with a typical molecularweight greater than about 15,000 daltons), (2) chemical modificationwith 4-aminobutyl-dimethyl-methoxysilane, and/or (3) attachment ofmolecules that bind (specifically or nonspecifically) to the cellsurface (e.g., lectins), among others.

[0137] The fluid compartments comprise open regions of the substratesurface adjacent the aperture to which fluid is confined. Here, fluid isconfined by a combination of hydrophilic and hydrophobic interactions.Specifically, fluid is attracted to the region adjacent the aperture byhydrophilic interactions and excluded from regions away from theaperture by surrounding layers of hydrophobic material 168 a,b attachedor bound to the surface. Consequently, the buffer compartments aredelineated by the surface of the substrate on one side and by surfacetension on the opposing side, creating dome-shaped compartments, asshown.

[0138] The electrodes comprise conductive elements such as Ag/AgCl forgenerating an electric potential across the aperture. The electrodes,which may be used for positioning and/or recording, are immersed in thefluid compartments. The electrodes may be directly attached to thesubstrate (e.g., by sputtering or printing) or to a container thatcontains the substrate. Here, a first (measurement or recording)electrode is used to apply a measurement voltage, and a second(reference) electrode is used to apply a ground.

[0139] The measurement system may be used for positioning and/oranalyzing samples, at least substantially as described above. Inparticular, upon application of a voltage between the two fluidcompartments, mediated by the redox electrodes immersed in the twocompartments, a strongly inhomogeneous field is created around theaperture that attracts cells, vesicles, and other charged objectstowards the aperture. After these samples bind and/or form membranes,they may be analyzed electrically and/or optically, among others.

Example 5

[0140] Measurement System Having Multiple Measurement Sites

[0141] This example, illustrated in FIGS. 6 and 7, describes anexemplary measurement system 190 having multiple measurement sites, inaccordance with aspects of the invention. The drawings show twoalternative embodiments, separated by break lines, that includedifferent carrier/electrode configurations.

[0142] The measurement system includes a plurality of measurement sites,each capable of positioning and/or analyzing a sample, as describedabove. More specifically, the measurement system includes a substrate192, a plurality of fluid compartments 194 a,b, and a plurality ofelectrodes 196 a,b. The measurement sites are formed from portions ofthe substrate and combinations (e.g., pairs) of fluid compartments andelectrodes. The substrate preferably comprises (1) a silicon body 198,(2) a silicon nitride diaphragm 200 having a plurality of apertures 202,at least one per measurement site, and (3) a hydrophobic and/orinsulating surface coat 204. The fluid compartments preferably comprise(1) a plurality of measurement compartments 194 a, and (2) at least onereference compartment 194 b. The electrodes preferably comprise (1) aplurality of measurement electrodes 196 a, at least one per aperture ormeasurement site, and (2) at least one reference (ground) electrode 196b. The measurement system further may include additional features, suchas (1) a support or carrier plate 206 to simplify the design and/or toincrease the reliability of the system, and/or (2) one or more referencefiducials 207 for reference and/or alignment purposes, as describedbelow.

[0143] The components of the measurement system are described in moredetail in subsequent subsections. Briefly, on one side, the substratecontains a patterned surface that physically separates the measurementcompartments, allowing independent measurements. The patterned surfacemay be created by the patterned attachment of hydrophilic materials atmeasurement sites and hydrophobic materials at intervening positions.The measurement compartments may be accessed independently using aseparate measurement electrode for each compartment, where eachelectrode is connected independently to one or more voltage sources,such as a voltage clamp circuit. On the other side, the substratecontains a reference compartment that can be separated but thatpreferably is unified to form a single compartment in contact with asingle (usually ground) electrode. In some embodiments, the substrateincludes a silicon chip containing grooves that are closed by a siliconnitride/silicon oxide diaphragm. The diaphragm includes a small aperturehaving a diameter of less than about 20 μm. The substrate otherwise maybe surrounded by an insulating layer, for example, a thermally grownsilicon oxide layer, to reduce the system capacitance.

[0144] Substrate

[0145] The substrate generally comprises any structure adapted toprovide two or more sites for positioning and/or analyzing sampleselectrically, as described above. The substrate may be formed from anysuitable material, including silicon, plastic, and/or glass. Thesubstrate generally may include any number of sample sites arranged inany suitable format, as described above. Preferred formats include 8×12(96) rectangular arrays and 16×24 (384) rectangular arrays, withstandard microplate footprints. Some embodiments may include additionalsites, including additional rows or columns of sites. For example, inone such embodiment, the system includes an additional row of sites,configured as an 8×13 (104) rectangular array.

[0146] Fluid Compartments

[0147] The fluid compartments generally comprise any region adapted tosupport fluid for bathing the sample and for providing electricalcontact between the measurement and reference compartments.

[0148] The measurement compartments are used for positioning and/oranalyzing samples. These compartments are defined by hydrophilic spotson the chip surface, surrounded by a hydrophobic surface coating, forlocalizing fluid. The measurement compartments include an aperturepositioned within the hydrophilic spot and a measurement electrodepositioned for electrical contact with the associated measurement fluid.The hydrophilic spot typically includes an at least substantially planaror concave adhesion surface, which may be selected and/or treated asdescribed above to promote sample binding and/or membrane formation.

[0149] The reference compartments are used for completing the electriccircuit, typically to electric ground. These “backside” compartments maybe combined to form one or more large compartments (corresponding to twoor more measurement compartments), since the separate compartmentstypically would contain the same buffer solution and each be connectedto ground. In particular, a single large backside compartment and asingle backside electrode are sufficient for spatial resolution ofindividual recordings, if the measurement compartments are addressedindividually. In some embodiments, the backside electrode may bedeposited directly on the recording chip or on an embedding cartridge.

[0150] Electrodes

[0151] The electrodes generally comprise any mechanism adapted to applyand/or measure an electric potential across the aperture, with eachmeasurement site in contact with at least one measurement electrode andat least one reference electrode, as described above.

[0152] The composition of the electrodes is selected to allow currentflow at physiological potentials. Preferred electrodes includesilver/silver chloride (Ag/AgCl) and/or platinum (Pt) redox electrodesfor both the sample and reference compartments. Particularly preferredelectrodes include silver (Ag) as the electrode material, chlorinatedwithin a chlorine (Cl₂) atmosphere.

[0153] The number of electrodes may vary. The upper side of thesubstrate (associated with the measurement compartments) preferablyincludes enough separate electrodes independently to address eachcorresponding recording site, e.g., 8×13 electrodes on a 2.25 mm grid.In contrast, the lower side of the substrate (associated with thereference compartment) preferably includes a single electrode.

[0154] The electrodes may be insulated outside the measurementcompartment. Preferred insulation material preferably has a highelectrical resistance and a low dielectric constant and loss.Particularly preferred insulation material is produced from Teflon,silicon nitride (Si₃N₄), and/or Sylgard by spin coating or chemicalvapor deposition (CVD). These materials are sufficiently hydrophobic(even after short oxygen-plasma treatment) to confine the measurementand reference compartments. Moreover, these materials may include or beformed to include grooves or holes, potentially improving fluid supportand/or reducing evaporation.

[0155] The electrodes at the various measurement sites may be connectedelectrically to corresponding contacts 208 for clean and/or easy accessto appropriate electronic components, such as amplifiers, recordingdevices, and the like. In a preferred embodiment, the contacts arepositioned near the edge or border of the substrate, and a bonding wire210 joins the electrodes and contacts, although more generally anymechanism capable of establishing an electrical connection may beemployed. In particular, the electrodes can be bonded to contacts placedon a plastic (e.g., polypropylene) carrier that embeds the entirerecording chip.

[0156] Support Element

[0157] The support element generally comprises any mechanism forindependently and portably supporting the substrate and associatedsystem components, potentially simplifying design and/or increasingreliability. The support element may support the substrate at its edgesand/or in its interior, with the interior support potentially reducingor preventing sagging and/or stress of the substrate. In an exemplaryembodiment, the support element includes a carrier plate 206 and aspacer 212 sandwiched between the substrate and the carrier plate nearthe edges of the substrate. The carrier plate may be formed from glass(e.g., PYREX) and/or any other suitable material. The substrate, spacer,and carrier plate may be joined using any suitable mechanism, such asanodic bonding. The separation between the substrate and the carrierplate (i.e., the spacer thickness) preferably is chosen to be less thanabout 1 mm, to allow filling of the backside (i.e., reference)compartment by capillary forces. By extending the glass plate over theborders of the chip, in some embodiments, it may be possible to bond theupper electrodes to contacts placed on the glass plate.

[0158] Reference fiducials

[0159] The reference fiducials generally comprise any feature orcharacteristic of the measurement system adapted to provide informationthat facilitates sample handling and/or analysis, for example, asdescribed in U.S. Pat. No. 6,258,326, issued Jul. 10, 2001, which isincorporated herein by reference.

[0160] The reference fiducials, or a subset thereof, may be used toencode information and/or to provide reference positions. For example,the reference fiducials may encode information relating to the identityof the manufacturer of the system and/or one or more properties of thesystem and/or the associated samples. Alternatively, or in addition, thereference fiducials may provide reference positions useful to correctfor cross-system drift (due to dimensional irregularities in the system)and/or to align the system with ancillary devices, such as an electricaldevice for electrical analysis and/or an optical device for opticalanalysis.

[0161] The reference fiducials may encode information using any suitablemechanism, including electrical and/or optical mechanisms. For example,the reference fiducials may encode information electrically, based onthe resistance, capacitance, and/or inductance, among others, of aparticular portion or portions of the system. Alternatively, or inaddition, the reference fiducials may encode information optically,based on the size, shape, position, color, absorptivity, reflectivity,and/or transmissivity of a particular portion or portions of the system.

[0162] The reference fiducials may be identified and read using anysuitable mechanism, including the electrical device and/or opticaldevice used in sample analysis.

Example 6

[0163] Measurement System Having Optical Measurement Aids

[0164] This example, illustrated in FIG. 8, describes an exemplarymeasurement system 230 having optical measurement aids, in accordancewith aspects of the invention.

[0165] The measurement system includes a substrate 232, a plurality offluid compartments 234 a,b, a plurality of electrodes 236 a,b, and acarrier plate 238, at least substantially as described above in Example5. However, the system further includes an optical measurement aid foruse in conjunction with a suitable optical analysis system, as describedabove. The optical measurement aid generally comprises any element ormechanism adapted to facilitate and/or enable optical analysis ofsamples such as cells or vesicles positioned on or near the substrate.The optical measurement aid may comprise a modification of one or moreof the elements listed above and/or a new element in addition to and/orin lieu of one or more of the elements listed above.

[0166] The optical measurement aid may comprise a support element thatincludes a short spacer and/or a thin, optically transparent carrierplate, as described above. A short spacer and/or a thin carrier platemay shorten the optical path length between the optical device andsample, by reducing the separation between the substrate and carrierplate. A thin carrier plate also may better match the opticalrequirements of the optical analysis system. To this end, the thicknessof the carrier plate may be selected to correspond to the thickness of astandard microscope cover slip, for example, 0.08 to 0.13 mm thick (No.0), 0.13 to 0.17 mm thick (No. 1), 0.16 to 0.19 mm thick (No. 1½), or0.17 to 0.25 mm thick (No. 2), among others. To related ends, thecarrier plate may be selected to improve overall optical transmission,for example, by using crystal-clear, pure water-white glass or superclarity, clear-white borosilicate glass. Alternatively, or in addition,the carrier plate may be selected to improve transmission of polarizedlight, for example, by using strain-free glass or fused silica.Alternatively, or in addition, the carrier plate may be selected to haveuniform surface quality, exceptional flatness, and/or precisedimensions, among others.

[0167] The optical measurement aid also may comprise a carrier plate orother interface having an array of lenses 240 such as microlenses thatcorrespond in number and spacing to the array of measurement sites.These lenses may be formed from any suitable material (such as glass orplastic) using any suitable technique (such as etching or molding). Thelenses may be used for high-magnification and/or high numerical apertureanalysis of samples, including the analysis of single cells positionedon single apertures. To assist such analysis, the x-y resolution of therecording apertures and microlenses preferably is less than a few (e.g.,about 1-4) μm, which may be facilitated using a high-precision bondingprocess. Moreover, the z resolution of these components preferably isless than a few (e.g., about 1-4) μm, which may be facilitated usinglenses having high numerical apertures.

[0168] The lenses in the array generally may have any shape capable ofcollecting light from the sample and/or focusing light onto the sample.For example, the lenses may be plano-convex, meaning that they have aflat (plano) surface and an opposed outwardly bulging (convex) surface.The plano-convex lenses may have two orientations. In the firstorientation, exemplified by lens 240, the convex surface 242 facestoward the sample site, and the planar surface 244 faces away from thesample site. In the second orientation, exemplified in FIG. 8 by lens240′, the convex surface 242′ faces away from the sample site, and theplanar surface 244′ faces toward the sample site. In either orientation,the lens will collect light transmitted from the sample and direct thecollected light toward a detector, such as an imaging detector (e.g., acharge-coupled device (CCD)) or a point detector (e.g., aphotomultiplier tube (PMT)), among others.

[0169] The optical measurement aid also may include a window 246 in thesubstrate having a shape configured to match an optical sensed volume,including the frustoconical shape through which excitation light isdirected onto the sample and/or from which emission light is detectedfrom the sample, for example, as described in U.S. patent applicationSer. No. 09/478,819, filed Jan. 5, 2000, which is incorporated herein byreference. The matching may be used in optical analysis to increasesensitivity (for example, by avoiding detection from walls of the samplewell) and/or to decrease sample volume, among others.

Example 7

[0170] Analysis System

[0171] This example, illustrated in FIG. 9, describes an exemplarymeasurement system 270 for combined electrical/optical detection, inaccordance with aspects of the invention.

[0172] The combined measurement system includes an electrical analysissystem 272 and an optical analysis system 274.

[0173] The electrical analysis system generally comprises any system forperforming electrical measurements such as patch clamp measurements on asample such as a cell, vesicle, or biological organelle. The electricalanalysis system may include any suitable combination of apertures 275,substrates 276, fluid compartments 278 a,b, and electrodes (not visiblein this view), among other elements, as described above. Exemplarysystems are described above in Examples 2-6.

[0174] The optical analysis system generally comprises any system fordetecting light transmitted from the sample, particularlyphotoluminescence and chemiluminescence light. The optical analysissystem may include a light source 280, a detector 282, and an opticalrelay structure 284 for transmitting excitation light from the lightsource to the sample and emission light from the sample to the detector.The system further may include additional components for performingadditional and/or duplicative functions, including (1) filters 286positioned in the excitation and/or emission optical paths for alteringthe intensity, wavelength, and/or polarization of the excitation andemission light, respectively, (2) confocal optics elements 288 such asan aperture positioned in an image plane for reducing detection ofout-of-focus light, and (3) a reference monitor 290 positioned to detecta portion of the excitation light for correcting for variations (e.g.,fluctuations and/or inhomogeneities) in the excitation light.

[0175] The light source generally comprises any mechanism for producinglight suitable for use in an optical assay, such as a photoluminescence,scattering, and/or absorbance assay, among others. Suitable lightsources include lasers, arc lamps, incandescent lamps, fluorescentlamps, electroluminescent devices, laser diodes, and light-emittingdiodes (LEDs), among others. The light source may be capable of use inone or more illumination modes, including continuous and time-varyingmodes, among others.

[0176] The detector generally comprises any mechanism for detectinglight transmitted from a sample in an optical assay. Suitable detectorsinclude charge-coupled devices (CCDs), intensified charge-coupleddevices (ICCDs), videcon tubes, photomultiplier tubes (PMTs),photodiodes, and avalanche photodiodes, among others. The detector maybe capable of use in one or more detection modes, including (a) imagingand point-reading modes, (b) discrete (e.g., photon-counting) and analog(e.g., current-integration) modes, and (c) steady-state andtime-resolved modes, among others.

[0177] The optical relay structure generally comprises any mechanism fortransmitting light between the light source, sample, and detector (orsimply the sample and detector in a chemiluminescence assay). Suitableoptical relay structures may include mirrors, lenses, and/or fiberoptics, among others. Here, the optical relay structure includes abeamsplitter that generally transmits excitation light toward the sampleand generally reflects emission light toward the detector.

[0178]FIG. 9 shows an exemplary embodiment of a combinedelectrical/optical measurement system, including components as describedabove. Here, a parallel read-out system is used for confocal-opticalrecordings, for example, using the chip substrate system of FIG. 8. Ineither order, the chip substrate is placed in an appropriate light beamthat is able to excite fluorescent probes of interest, and the samplemembranes or cells are positioned at the individual apertures of thechip. The samples are excited using one parallel light beam, forexample, using a 45-degree mirrored beamsplitter. The fluorescent lightcoming from the biological sample or any associated fluorescent probesis transmitted to an optional filter and confocal optics element toincrease the signal-to-noise ratio before being projected onto the lightsensitive chip of a CCD camera. The confocal optics element reduces oreliminates out-of-focus light not originating from the sample. Thespatial resolution of the CCD chip allows detection of fluorescence fromall apertures (and consequently all biological samples) simultaneously,if desired.

[0179] More generally, the system may be configured to allow top and/orbottom illumination and/or detection of the sample(s), permitting thefollowing combinations: (1) top illumination and top detection, or (2)top illumination and bottom detection, or (3) bottom illumination andtop detection, or (4) bottom illumination and bottom detection.Same-side illumination and detection, (1) and (4), is referred to as“epi” and is preferred for photoluminescence and scattering assays.Opposite-side illumination and detection, (2) and (3), is referred to as“trans” and is preferred for absorbance assays.

[0180] Alternatively, or in addition, the system may be configured toallow illumination and/or detection at oblique angles. For example,illumination light may impinge on the bottom of a sample holder at anacute angle (e.g., about 45 degrees) relative to detection. Incomparison with a straight-on epi system (light source and detectordirected at about 90 degrees to sample holder) or a straight-throughtrans system (light source directed through sample holder directly atdetector), an oblique system may reduce the amount of excitation lightreaching the detector.

[0181] Suitable systems, and components thereof, for top/bottom and/oroblique illumination are described in the following materials, which areincorporated herein by reference: U.S. Pat. No. 5,355,215, issued Oct.11, 1994; U.S. Pat. No. 6,097,025, issued Aug. 1, 2000; and U.S.Provisional Patent Application Serial No. 60/267,639, filed Aug. Feb.10, 2001.

Example 8

[0182] Producing, Sizing, and Binding of Vesicles

[0183] This example describes exemplary methods for producing, sizing,and binding lipid vesicles.

[0184] A mixture of 100 μL asolectin (Fluka) or egg lecithin (EPC), 50μL palmitoyloleylphosphatidylglycerol (POPG), and 3 μL dipalmitoylphosphatidyl-ethanolamine-rhodamine (DPPE-rhodamine) (Molecular Probes,USA) (all 10 mg/mL in chloroform, Avanti Polar Lipids), and 70 μLmethanol is dried in a rotary vaporizer (Büchi Rotavapor R-114) at low(400 mm Hg) pressure in a 10 mL round flask to form a film. After a1-hour incubation under vacuum, to the flask is added either 10 mL H₂Oor 10 mL of a buffer solution having a concentration of less than 150 mMof KCl and/or less than 600 mM of sucrose or preferably of sorbitol.After a subsequent 16-hour incubation at 37° C., the resulting lipidvesicles appear as an almost transparent cloud. The lipid vesicles areaspirated and removed using a 1 mL pipette and stored at 4° C. untilfurther use. Storage of the vesicle solution may be improved by theaddition of sodium azide (NaN₃) to a concentration of 0.2% by weight.This vesicle preparation procedure yields mostly (>90%) unilamellarvesicles, with sizes up to 250 μm. However, some of the vesicles maycontain additional smaller vesicles, which are not relevant for membraneformation.

[0185] The ability of the resulting vesicles to establish electricallytight seals against a surface aperture is enhanced by purification ofthe initial vesicle mixture to remove vesicles and lipid impurities thatare smaller than about 10 μm in size. Without such purification, thebinding of such smaller vesicles in the vicinity of the aperture mayprevent electrically tight sealing of the aperture by large (e.g.,larger than 10 μm) vesicles. The vesicles may be sized by dialysis, forexample, using a nylon mesh with a 20-μm pore size for at least about 20hours. If necessary, the membrane fluidity of the resulting vesicles maybe lowered, for example, by adjusting the lipid composition so that itincludes a higher fraction of low-fluidity lipids and/or by lowering thetemperature so that it is closer to the phase-transition temperature ofthe vesicles (e.g., less than or equal to about 4° C., or morepreferably less than or equal to about 1° C., for some lipids). Theunilamellarity of the resulting vesicle membranes may be demonstratedand/or verified using any suitable analytical technique, such asmicroscopic analysis using a confocal microscope.^(xi)

[0186] More generally, vesicles may be produced, sized, and bound usingany suitable methods. For example, large unilamellar vesicles (giantunilamellar vesicles, GUVs) may be produced using the hydrationmethod.^(xii) Similarly, proteoliposomes may be produced using anappropriately modified hydration method.^(xiii) Additional vesicles maybe produced using the methods described in U.S. patent application Ser.No. ______ , filed Sep. 14, 2001, titled EFFICIENT METHODS FOR THEANALYSIS OF ION CHANNEL PROTEINS, and naming Christian Schmidt asinventor.

Example 9

[0187] Electrophoretic Positioning of Vesicles

[0188] This example, illustrated in FIG. 10, describes exemplary methodsfor electrophoretically positioning samples such as cells and lipidvesicles. The positioning attainable using these methods may exceed thatattainable using gravity sedimentation, in at least the following ways:(1) a decrease in the necessary number of vesicles or cells, (2) anincrease in the total rate of membrane formation, and (3) an increase inthe probability of a successful membrane setup or cell binding. Vesiclesand cells also may be prepositioned prior to electrophoretic positioningto improve performance, for example, by introducing the vesicles orcells to the sample so that they are initially positioned above theaperture.

[0189] The electrophoretic positioning methods make use ofinhomogeneities in the electric potential and associated electric fieldsurrounding the aperture. FIG. 10 shows results of a finite elementmethod (FEM) simulation of the electric potential distribution around asubstrate in accordance with aspects of the invention. The substrateincludes a 4-μm aperture positioned between parallel electrodes. Thefield distribution is shown as a series of equipotential lines(corresponding a cross-section through the three-dimensionalequipotential surfaces), with a spacing of 4 mV, where the potentialdifference between the electrodes is 80 mV. The field-line curve isdistorted in this simulation from its normal circular form to anelliptical form to reflect leak currents in the edge region of thecarrier chips. The following parameters were used in the simulation:C_(buffer)=10 mM KCl, V=80 mV, d_(aperture)=4 μm, and spacing betweenthe aperture and each electrode=1 mm.

[0190] The electric field associated with an electric potential is minusthe spatial rate of change of the potential, i.e., E=−∇∇. Thus, theelectric field is perpendicular to the equipotential surfaces at allpositions, pointing in the direction of decreasing potential. Moreover,the electric field is stronger where the equipotential surfaces arecloser together, and weaker where these surfaces are farther apart.Consequently, from FIG. 10, the electric field points toward theaperture (on one side of the aperture), with a strength that increaseswith proximity to the aperture.

[0191] These electrophoretic positioning methods generally may be usedin any system capable of creating and focusing an electric field throughan aperture. In preferred systems, the electrodes used to create thefield are positioned relatively close together, for example, withinabout 5-10 mm, reducing the voltage required to create an acceptableelectric field. Specifically, the measurement and reference electrodesare located, one above and one below the substrate, at a distance ofabout 0.2 to 3 mm, preferably about 0.5-2 mm, and more preferably about0.5 to 1 mm. The clamp voltage generated by these electrodes is notcritical; however, it customarily lies in the range V_(c)=−300 to -300mV, preferably lies in the range−60 to -100 mV, and more preferably liesin the range−60 to −80 mV. The associated electrophoretic-driving forcedirects vesicles and cells, following the electric field, toward theaperture. In particular, because the electric field is stronglyinhomogeneous, increasing sharply in magnitude with proximity to theaperture, vesicles and cells move automatically toward the aperture. Inparticular, the fields are most effective near the aperture (e.g.,within about 200 μm of the aperture), so that samples preferably arebrought into this range by prepositioning or reach it convectively. Forthis purpose, a hole (for example, d<1 mm) may be located in themeasurement electrode with respect to the aperture.

[0192] The following subsections describe two alternative methods ofelectrophoretic positioning.

[0193] Variation 1

[0194] The offset voltage V_(offset) between the electrodes may becorrected before each measurement. To do so, 5 μL of buffer solution isadded directly to the aperture, and the measurement electrode is broughtto within about 1 mm from the substrate surface. After a liquid meniscusforms between the surface of the substrate and the electrode, the offsetvoltage and the capacitance of the system are adjusted to compensate.

[0195] A 10-μL dispersion of lipid vesicles subsequently is added to theupper side of the measurement electrode, where the vesicles can sedimentthrough the circular opening located in the measurement electrode.Vesicles that move through the measurement electrode opening may beaccelerated directly to the aperture opening under the influence of anelectric field generated by the applied electrode voltage, V_(M)=−50 to−80 mV. In doing so, the focusing achieved, measured in the number ofvesicles passing through the aperture opening with unmodified surfaces,is a function of the size of the window (that is, the portion of theSiO₂ layer laid open by etching). Smaller SiO₂ windows (e.g., less thanabout 45×45 μm²) clearly increase vesicle throughput.

[0196] Variation 2:

[0197] The offset voltage V_(offset) between the electrodes may becorrected before each measurement. To do so, 5 μL of buffer solution isadded between the substrate and the measurement electrode, oralternatively between the substrate and the reference electrode, afterwhich the voltage is determined at which the current flow vanishes,satisfying the expression I(V_(offset))=0.

[0198] A 3-μL dispersion of lipid vesicles subsequently is added to themeasurement compartment near the aperture, where, in the case of a planeparallel electrode arrangement, the vesicles can sediment through thecircular opening located in the measurement electrode. Vesicles thatcome into the vicinity (e.g., less than about 200 μm) of the apertureexperience a very high field intensity (generally but not necessarilybetween about 100 kV/m and several kV/m) and are accelerated accordingto the electric field curve directly to the aperture. After the vesiclesbind to the substrate and form an electrically tight seal, they areanalyzed electrically.

Example 10

[0199] Sealing of Vesicles with Unmodified Surfaces

[0200] The example, illustrated in FIG. 11, describes the sealing ofvesicles, as described above, with unmodified surfaces. In particular,FIG. 11 shows a plot of current versus time after the docking or bindingof vesicles to a 7-μm aperture in the unmodified surface of a suitablesubstrate. The plot shows that the addition of Ca²⁺ to a finalconcentration of 4 mM leads quickly to a tight electrical seal betweenthe vesicle membrane and the substrate surface. Specifically, theaddition of Ca²⁺ causes a rapid, significant drop in current, indicatingthat the membrane has at least substantially blocked ion pathwaysthrough the aperture.

Example 11

[0201] Binding and Adsorption of Vesicles on Modified SiO₂ Surfaces

[0202] The example, illustrated in FIG. 12AB, describes the bindingand/or other interactions of vesicles as described above withpolylysine-modified SiO₂ surfaces.

[0203] The binding between these vesicles and surfaces may be strong andrapid, manifesting itself in less than about 0.5 seconds after anappropriate proximity is reached. The probability of successfullypositioning a vesicle and subsequently forming an electrically tightmembrane seal is strongly dependent on the size of the aperture, thesize of the SiO₂ window, and the number, size, and size distribution ofthe vesicles in the vesicle solution. If substrates having aperturediameters less than about 2 μm and window sizes greater than about 40 μmare used in conjunction with suspensions of vesicles having a vesiclediameter greater than about 40 μm, the probability of binding andforming an electrically tight seal may exceed 90% (n>15, where n is thenumber of trials). In general, a decrease in the width of the apertureand an increase in the purity of the vesicle suspensions lead to greaterreproducibility in the formation of tight aperture seals, both substrateto substrate and vesicle preparation to vesicle preparation.

[0204] Vesicles that bind to the surface subsequently may be drawn outto form substantially flat, defect-free membranes. Fluorescencemicroscopy studies performed using vesicles labeled with both rhodamine(to label vesicle membranes) and carboxyfluorescein (to label vesicleinteriors) show that vesicles flatten and may burst upon binding,forming unilamellar structures, since bound vesicles appear flat andred, suggesting that carboxyfluorescein has been released. These studieswere conducted using polylysine-coated glass and a confocal microscope(LSM 510, Zeiss Jena, Germany). Electrical studies of membraneresistance, R_(M), demonstrate that the bound vesicles may formsubstantially defect-free lipid membranes, since very high membraneresistances (e.g., R_(M)>6.4 GΩ (n=26)) are measured on the substratesin symmetric 85 mM KCl. An analogous series of measurements in symmetric10 mM KCl demonstrates binding of the vesicles, after appropriateproximity, in less than about 0.2 seconds, with a probability greaterthan about 70% (n>15) and a membrane resistance greater than about 10GΩ.

[0205] To promote strong adhesion of the vesicles, the surface of thesubstrate, in given cases, is coated with an adhesion promoter, forexample, polycations.^(xiv) For physisorption, for example, an aqueoussolution of polycations (e.g., 0.1% poly-1-lysine bromide, MW 100,000,Sigma) may be added to the substrate for about 2-5 minutes directlybefore the measurement and subsequently rinsed off with measurementbuffer solution. The covalent binding of peptide polycations preferablyis done using previously activated hydroxyl groups of the quartzsurface, for example by means of tosyl chloride(triphenylchloromethane).^(xv) Through the modification of the substratesurface, an attraction of vesicles with negative surface charge isachieved, which is completely sufficient for electrically tight sealsbetween the membrane and the substrate surface. Alternatively, thesurface also may be modified by other compounds having cationcharacteristics in the desired pH range, such as, for example,4-aminobutyl-dimethyl-methoxysilane. Finally, treating the substrates inO₂ plasma for several minutes before surface modification leads to moreconsistent surface characteristics.

[0206]FIG. 12AB shows the time course of vesicle binding and thesubsequent development of membranes with very high electrical insulationresistance for a 4-μm aperture (FIG. 12A) and a 7-μm aperture (FIG. 12B)in a poly-L-lysine-coated SiO₂ substrate. The measurements wereperformed in the presence of 10 μM KCl, using a clamp voltage of −80 mV.

Example 12

[0207] Membrane Electrical Properties

[0208] This example describes electrical factors relating to preferredaperture sizes, including characteristic electrical properties.

[0209] The thermal noise σ of a circular lipid membrane is proportionalto R_(M) ^(−½):^(xvi) $\sigma = \sqrt{\frac{4{kTf}_{c}}{R_{M}}}$

[0210] where R_(M)=R_(spec)/(πr_(M) ²). It follows therefrom that$\sigma = {r_{M}\sqrt{\frac{4\pi \quad {kTf}_{c}}{R_{spec}}}}$

[0211] In these formulae, σ is the effective noise flow, r is theradius, f is the frequency, k is the Boltzmann constant, R is theresistance, and T is the temperature.

[0212] Thus, to be a usable membrane for measurement purposes,r_(M)/{square root}{square root over (R_(spec))} should be very small.The minimization of this product can be pursued according to theinvention in two ways: (1) by minimizing the membrane radius r_(M),and/or (2) by the electrically tight sealing of the membranes used.

Example 13

[0213] Electrical Parameters of Lipid Membranes

[0214] This example describes the effects of vesicle fusion onelectrical parameters of the measurement system, as described above.

[0215] The resistance across the sample substrate changed significantlyfollowing vesicle fusion. Before fusion, the resistance is up to 1 MΩ(usually <450 kΩ) in 85 mM KCl, and similarly usually up to 1 MΩ in 1 mMKCl, depending in both cases on the size of the aperture. Greaterresistances are interpreted as artifacts, possibly reflecting, forexample, the inclusion of air bubbles under the aperture opening. Afterfusion, the resistance is greater than about 6.4 GΩ in 85 mM KCl,greater than about 10 GΩ in 10 mM KCl, and greater than about 40 GΩ in 1mM KCl, corresponding to four order-of-magnitude increases inresistance. Here, the resistance R is at least approximately related tothe applied voltage V and the current I according to Ohm's law, i.e.,R=V/I.

[0216] The capacitance of the sample substrate changed onlyinsignificantly following fusion, by several pF in 85 mM KCl, and by 160to 280 pF in 1-10 mM KCl.

Example 14

[0217] Vesicle Passage through Micrometer Pores

[0218] This example, illustrated in FIG. 13, describes the passage ofvesicles through apertures in the substrate.

[0219] The passage of vesicles through an aperture in the presence ofnegatively charged surfaces, such as unmodified SiO₂ layers, can beobserved by monitoring changes in current (or, equivalently, resistance)across the aperture. Specifically, the passage of vesicles will lead toa decrease in current and an associated increase in resistance. Tomonitor for artifacts in the observed values, the polarity of thevoltage is reversed, whereupon no modulation of current or resistance isobserved.

[0220] The duration of observed changes in the amplitude of resistancecan be correlated with size of the vesicles passing through the aperturebeing monitored. For example, modulations in the amplitude of resistancelasting up to 18 seconds suggest the passage of very large vesicles withsufficiently fluid membranes. Where vesicle populations with diametersgreater than about 50 μm (n=4) are used in conjunction with apertureopenings with diameters of about 7 μm, an almost exclusive variation ofthe time of passage for fixed changes in amplitude as a function ofvesicle size is observed. It is presumed that vesicles undergoingpassage through the aperture opening are drawn out during their passageto form tubular structures with definite diameters and closed surfaces.

[0221] By analyzing the typical time of passage for large vesicles(d_(vesicle)>>d_(aperture)) and the typical change in the amplitude ofresistance for small vesicles (d_(vesicle)˜d_(aperture)), thecomposition of the vesicles with respect to size can be determined for agiven vesicle solution. The method of the invention therefore possessesutility for analyzing size distributions in selected populations ofvesicles and cells.

Example 15

[0222] Observation of Alamethicin Pores and Nicotinic AcetylcholineReceptors

[0223] This example, illustrated in FIGS. 14 and 15AB, describesobservation of electrical activity of alamethicin pores and nicotinicacetylcholine receptors (nAChR) in fused vesicles. These observationsconfirm the biological utility of the invention.

[0224]FIG. 14 shows a plot of current versus time for membranescontaining alamethicin. In these experiments, a membrane is formed overan aperture in 85 mM KCl. Next, alamethicin is added to the measurementcompartment, to a final concentration of 0.1 μg/mL.^(xvii) Finally, apotential is applied, and a plot of current versus time is generated.The amplitude and dwell times of the current fluctuations observed inthis plot, corresponding to 600 pS conductivities of the alamethicinpores, prove the functionality and high sensitivity of the system.

[0225]FIG. 15AB shows an analogous plot of current versus time formembranes containing the membrane protein nAChR. In these experiments, amembrane is formed as above. Next, nAChR is introduced into the membranevia Ca²⁺-mediated fusion. Specifically, nAChR is purified from anappropriate source, and then reconstituted into small unilamellarvesicles.^(xviii) Next, these vesicles are added to the measurementcompartment, and then fused to the membrane by increasing the Ca²⁺concentration of the sample chamber to greater than about 1 mM. In givencases, the fusion is supported by the subsequent temporary setup of anosmotic gradient.^(xix) Finally, a potential is applied, and a plot ofcurrent versus time is generated. In the absence of agonists, typicalreceptor opening events are observed (FIG. 15A), whereas, in thepresence of agonists, such as carbamylcholine (20 μM finalconcentration), such receptor opening events are substantiallyextinguished within a short time (t<100 seconds) (FIG. 15B).

Example 16

[0226] Analysis of Cells

[0227] This example, illustrated in FIGS. 16-18AB, describes use of theabove-described systems in the study of cells.

[0228] The methods of the invention are generally applicable to theinvestigation and analysis of cells. Such cells may be positioned andelectrically characterized using procedures substantially analogous tothose described above for vesicles. However, in some cases, thepositioning and measurement methods may be modified as desired toaccommodate differences in vesicles and cells, including, among others,the cytoskeleton, the varied lipid and protein content of cellmembranes, and the cell wall in plants and certain algae, bacteria, andfungi. For example, the cell may be made more flexible by disrupting thecytoskeleton, for example, using cytochalasin and/or colchicine.Similarly, in measurements using plant cells, the cell wall may beremoved to expose a relatively smooth membrane surface capable offorming a tighter electrical seal. Similarly, in measurements usinganimal cells derived from tissues, the extracellular matrix may beremoved or digested, for example, using one or more proteases, lipases,and/or glycosidases, among others.

[0229] The methods may be used for a variety of patch clamp experiments,in a variety of formats or configurations. The cell, as initially boundand sealed, is in a “cell-attached configuration.” If the membrane patchover the aperture then is ruptured or destroyed, for example, byapplying a pulse of voltage or suction, electrical measurements can beperformed over the entire cell membrane in a “whole-cell configuration.”Alternatively, if the membrane patch over the aperture is permeabilized,for example, by the addition of pore formers such as amphotericin B ornystatin to the reference compartment, electrical measurements again maybe performed over the entire cell membrane in a “perforated-patchconfiguration.” Alternatively, if the cell (instead of the membranepatch over the aperture) is lysed, electrical properties of the patchmay be measured in an “inside-out configuration.” In the lattermostapproach, the cytosolic side of the membrane is exposed to themeasurement solution, and the relatively small area of membrane beinganalyzed potentially makes possible the study of individual channelevents.

[0230] FIGS. 16-18 show exemplary results of positioning and voltageclamp experiments performed using Jurkat cells. These cells, a humanmature leukemic cell line, phenotypically resemble resting human Tlymphocytes and are widely used to study T cell physiology. Similarresults (not shown) were obtained using TE 671 cells and CHO cells.

[0231] The cells were cultured and prepared using standard cell culturetechniques. These cells were maintained for 2-5 days at 37° C. in 5% CO2in RPMI with Glutamax, supplemented with 10% FCS and P/S (100 U/100μg/mL). Before use, cells were resuspended in a physiological buffer(PB=NaCl, 140 mM; HEPES, 10 mM; KCl, 5 mM; CaCl₂, 2 mM; MgCl₂, 1.2 mM;pH 7.3, osmolarity 290 mOsm) at a density of 10⁷ cells/mL. Lower andupper fluid compartments were filled with 20 μL and 15 μL of PB,respectively. Five μL of the cell suspension was added to the uppercompartment. Positioning was made at V_(m)=−60 or −90 mV. Allexperiments were performed at room temperature.

[0232]FIG. 16 shows the time course of positioning, binding, andsubsequent development of a tight electrical seal for a Jurkat cell. Thecell was positioned at −60 mV. Seal formation occurred about 15 secondsafter cell addition, quickly rising to about 1000 GΩ. The aperture sizewas about 3 μm, and the chip resistance was about 250 kΩ.

[0233]FIG. 17 shows a series of plots of current versus time showing thecurrent flowing through the membrane of a Jurkat cell at the indicatedpositive and negative clamp voltages in a cell-attached configuration.The curves appear quantized, switching largely between just two values,one low and one high, particularly at higher voltages. Thischaracteristic suggests that single-channel events are being observed,corresponding to the opening and closing of the channel.

[0234]FIG. 18 shows an analysis of the current flowing through themembrane of a Jurkat cell for a +60 mV clamp voltage. Panel A shows arepresentative plot of current versus time. This plot again has aquantized character, like FIG. 17. Panel B shows a histogram plottingthe relative occurrence of a given current versus the current. Thehistogram is bimodal, with peak values of about 1.3 pA (the relativelysmaller peak at left) and about 4.8 pA (the relatively larger peak atright), corresponding to an average of about 3.5 pA.

Example 17

[0235] Miscellaneous Applications

[0236] The positioning and measurement systems provided by the inventionmay be used for a variety of purposes and a variety of assays. Exemplarymiscellaneous applications are described below.

[0237] Screening of Ingredients The system may be used to screenlibraries according to any suitable criterion, such as theidentification of candidate drugs, modulators, and the like. Suitablelibraries include compound libraries, combinatorial chemistry libraries,gene libraries, phage libraries, and the like. The system isexceptionally well suited to probing libraries whose members are presentonly in small amounts, such as (1) the large number of potential ligandsthat can be produced using combinatorial chemistry, and (2) manyreceptor proteins, above all ligand-controlled and G-protein-coupledreceptors (GPCRs). Owing to the process according to the invention, oralternatively the measurement arrangement/measurement apparatusaccording to the invention, it is possible to work with very few cells,either directly or after previous isolation and reconstitution of thereceptor proteins in vesicles or lipid membranes. By the uncomplicatedarrangements of the sensor elements in arrays, different substances orreceptors can be selected simultaneously. There is moreover thepossibility of receptor cleaning and reconstitution in lipid vesiclesmicrochromatographically in on-chip containers that optionally may beintegrated into the apparatus according to the invention.

[0238] Replacement of Conventional Patch-clamp Technologies

[0239] Conventional patch-clamp technologies form the foundation of theinvestigation of the functionality of membrane receptors as well as themodification of membrane characteristics as a response to signal andmetabolic processes in cells. If isolated cells of a homogeneous cellpopulation serve as the object of the investigation, as is, for example,often the case in transformed cells, the process according to theinvention serves as an at least comparable replacement for thepatch-clamp technologies. As objects of investigation for this process,for example, dissociated neurons and cultivated mammalian cells as wellas plant protoplasts are suitable.

[0240] Portable Biosensors/Environmental Analytics

[0241] The automation and outstanding mechanical stability of themeasurement system according to the invention permits its use inbiosensors. By using suitable transformed cells, receptors reconstitutedin vesicles, or channel-forming proteins, sensors can be set up that aresensitive to very different substrates or metabolites. Moreover, ifsufficiently tight electric seals are formed, which is possible usingthe apparatus according to the invention, then measurement sensitivitywill in principle only be dependent on the binding constant of thereceptor. This sensitivity may lie under one nanomole forG-protein-coupled receptors, and in the nanomolar range for ionotropicreceptors (e.g., 5 HT3, nAChR, GABA_(A)R, glycine R, and GluR).^(xx)

[0242] The disclosure set forth above may encompass multiple distinctinventions with independent utility. Although each of these inventionshas been disclosed in its preferred form(s), the specific embodimentsthereof as disclosed and illustrated herein are not to be considered ina limiting sense, because numerous variations are possible. The subjectmatter of the inventions includes all novel and nonobvious combinationsand subcombinations of the various elements, features, functions, and/orproperties disclosed herein. The following claims particularly point outcertain combinations and subcombinations regarded as novel andnonobvious. Inventions embodied in other combinations andsubcombinations of features, functions, elements, and/or properties maybe claimed in applications claiming priority from this or a relatedapplication. Such claims, whether directed to a different invention orto the same invention, and whether broader, narrower, equal, ordifferent in scope to the original claims, also are regarded as includedwithin the subject matter of the inventions of the present disclosure.

[0243]^(i) J. P. Changeux (1993), “Chemical Signaling in the Brain,”Sci. Am. Nov. Pages 30 ff; A. G. Gilman (1995), Angew. Chem. Int. Ed.Engl. 34:1406-1428; M. Rodbell (1995), Angew. Chem. Int. Ed. Engl. 34:1420-1428.

[0244]^(ii) J. Hodgson (1992), Bio/Technology 9:973.

[0245]^(iii) J. Knowles (1997), “Medicines for the New MillenniumHunting Down Deseases [sic: Diseases].” Odyssey Vol. 3(1).

[0246]^(iv) O. P. Hamill, A. Marty, et al. (1981), “Improved Patch-clampTechniques for High-resolution Current Recording from Cells andCell-free Membrane Patches”. Pflugers Arch 391(2):85-100.

[0247]^(v) (Radler, J., H. Strey, et al. (1995), “Phenomenology andKinetics of Lipid Bilayer Spreading on Hydrophilic Surfaces.” Langmuir11(11):4539-4548.

[0248]^(vi) For example, polycations, such as described by Mazia,Schatten, et al. (see D. Mazia, G. Schatten et al. (1975), “Adhesion ofCells to Surfaces Coated with Polylysine.” J. Cell Biol. 66:198-200).

[0249]^(vii) J. Edelstein, O. Schaad, J. -P. Changeux (1997), “SingleBinding versus Single Channel Recordings: A New Approach to StudyIonotropic Receptors.” Biochemistry 36:13755-13760.

[0250]^(viii) See Hamill, Marty, et al. (1981), loc. cit.; J. G.Nicholls, A. R. Martin, et al. (1992), From Neuron to Brain: A Cellularand Molecular Approach to the Function of the Nervous System.Sunderland, Ma., Sinauer Associates, Inc.

[0251]^(ix) R. Horn, A. Marty (1988), “Muscarinic Activation of IonicCurrents Measured by a New Whole-cell Recording Method,” J. Gen.Physiol. 92(2):145-59; J. Rae, K. Cooper, et al. (1991), “Low AccessResistance Perforated Patch Recordings Using Amphotericin B.” J.Neurosci. Methods 37(1): 15-26.

[0252]^(x) Z. M. Pei, J. M. Ward, et al. (1996), “A Novel ChlorideChannel in Vicia Faba Guard Cell Vacuoles Activated by theSerine/Theonine Kinase, CDPK.” EMBO J. 15(23):6564-74.

[0253]^(xi) See, e.g., R. B. Gennis (1989), Biomembranes: MolecularStructure and Function. New York, Springer Verlag.

[0254]^(xii) H. H. Hub, U. Zimmerman, et al. (1982), “Preparation ofLarge Unilamellar Vesicles.” FEBS Lett. 140(2):254-256; P. Mueller, T.F. Chien, et al.(1983), “Formation and Properties of Cell-like LipidBilayer Vesicles.” Biophys. J. 44(3):375-81, K. Akashi, H. Miyata, etal. (1996), “Preparation of Giant Liposomes in Physiological Conditionsand their Characterization under an Optical Microscope.” Biophys. J.71(6):3242-50

[0255]^(xiii) M. Criado and B. U. Keller (1987), “A Membrane FusionStrategy for Single-channel Recordings of Membranes UsuallyNon-accessible to Patch-clamp Pipette Electrodes.” FEBS Lett.224(1):172-6; B. U. Keller, R. Hedrich, et al. (1988), “Single-channelRecordings of Reconstituted Ion Channel Proteins: An ImprovedTechnique.” Pflugers Arch 411(l):94-100.

[0256]^(xiv) See Mazia, Schatten, et al. loc. cit., 1975.

[0257]^(xv) M. L. Williamson, D. H. Atha, et al. (1989), “Anti-T2Monoclonal Antibody Immobilization on Quartz Fibers: Stability andRecognition of T2 Mycotoxin.” Analytical Letters 22(4):803-816.

[0258]^(xvi) B. Sakmann and E. Neher (1983), Single-channel Recording,New York, Plenum Press.

[0259]^(xvii) R. B. Gennis (1989), Biomembranes: Molecular Structure andFunction, New York, Springer Verlag.

[0260]^(xviii) T. Schürholz, J. Kehne, et al. (1992), “FunctionalReconstitution of the Nicotinic Acetylcholine Receptor by CHAPS DialysisDepends on the Concentration of Salt, Lipid, and Protein,” Biochemistry31(21):5067-77; Schürholz, T. (1996), “Critical Dependence of theSolubilization of Lipid Vesicles by the Detergent CHAPS on the LipidComposition. Functional Reconstitution of the Nicotinic AcetylcholineReceptor into Preformed Vesicles above the Critical MicellizationConcentration,” Biophys. Chem. 58(1-2):87-96).

[0261]^(xix) See Eray, Dogan, et al. (1995) loc. cit.

[0262]^(xx) R. A. North (1994), Ligand- and Voltage-gated Ion Channels,CRC Press; S. J. Peroutka, (1991), Serotonin Receptor Subtypes-Basic andClinical Aspects, New York, John Wiley; Peroutka, S. J. (1994),G-protein-coupled Receptors, CRC Press; E. C. Conley, (1996), The IonChannel Facts Book, Academic Press.

I claim:
 1. A system for positioning and/or analyzing samples such as cells, vesicles, and cellular organelles, and fragments, derivatives, and mixtures thereof, comprising: a substrate having a plurality of apertures; at least one recording fluid compartment and at least one reference fluid compartment, arranged on opposites side of the substrate, and in contact via the apertures; and at least one recording electrode and at least one reference electrode, each in contact with at least one of the fluid compartments, and adapted to apply and/or measure an electrical potential across the apertures.
 2. The system of claim 1, further comprising a support element for independently supporting the substrate and associated system components.
 3. The system of claim 2, where the support element includes a carrier plate and at least one spacer sandwiched between the substrate and the carrier plate.
 4. The system of claim 3, where the space defined between the substrate and the carrier plate includes at least one of the compartments.
 5. The system of claim 3, where the carrier plate is made of a transparent material, such as glass, and includes a plurality of microlenses arranged in such a way that they allow the parallel optical observation of the samples that are positioned near the apertures.
 6. The system of claim 2, where each recording electrode contacts the substrate or the support element at least substantially at its border.
 7. The system of claim 1, where at least two fluid compartments are arranged on one side of the substrate, each of those compartments being in contact, via the apertures, with a single fluid compartment arranged on the other side of the substrate.
 8. The system of claim 1, where at least one electrode is arranged adjacent the substrate.
 9. The system of claim 1, where the recording fluid compartment is arranged above or below the substrate.
 10. The system of claim 1, where the substrate includes at least one material selected from the group consisting of silicon, silicon derivatives, glass, and plastic.
 11. The system of claim 1, where the diameter of the aperture is less than about 15 nm.
 12. The system of claim 11, where the diameter of the aperture is between about 0.3 μm and about 7 μm.
 13. The system of claim 1, where the number of recording fluid compartments is selected from the group consisting of 96, 384, 1536, and
 9600. 